Toll-like receptor-7 and -8 modulatory 1h imidazoquinoline derived compounds

ABSTRACT

The present disclosure provides novel imidazoquinoline derived compounds, derivatives thereof, analogues thereof, and pharmaceutically acceptable salts thereof, and methods of making and using such compounds. The present disclosure also provides TLR7 agonists and TLR7/TLR8 dual agonists, probes, tissue-specific molecules, adjuvants, immunogenic compositions, therapeutic compositions, and self-adjuvanting vaccines including the imidazoquinoline derived compounds, derivatives thereof, analogues thereof, and pharmaceutically acceptable salts thereof. Derivatives of the imidazoquinoline derived compounds also include dendrimers and dimers of the imidazoquinoline derived compounds, and methods of making and using the dendrimeric and dimeric imidazoquinoline derived compounds. The present disclosure also provides dual TLR2/TLR7 hybrid agonists that include imidazoquinoline derived compounds of the present disclosure.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. provisional applicationentitled “TOLL-LIKE RECEPTOR-7 AND -8 MODULATORY 1H IMIDAZOQUINOLINEDERIVATIVES,” having Ser. No. 61/487,320, filed on May 18, 2011, whichis entirely incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No.HHSN272200900033C awarded by the National Institute of Health (NIH) ofthe United States government. The government has certain rights in theinvention.

BACKGROUND

Toll-like receptors (TLRs) recognize specific molecular patterns presentin molecules that are broadly shared by pathogens, but are structurallydistinct from host molecules.^(1,2) The human genome includes 10 knownTLRs.² The ligands for these receptors are highly conserved microbialmolecules such as lipopolysaccharides (LPS) (recognized by TLR4),lipopeptides (TLR2 in combination with TLR1 or TLR6), flagellin (TLR5),single stranded RNA (TLR7 and TLR8), double stranded RNA (TLR3), CpGmotif-containing DNA (recognized by TLR9), and profilin present onuropathogenic bacteria (TLR 11).^(3,4) TLR1, -2, -4, -5, and -6 respondto extracellular stimuli, while TLR3, -7, -8 and -9 respond tointracytoplasmic PAMPs.² The activation of TLRs by their cognate ligandsleads to activation of innate immune effector mechanisms, including theproduction of pro-inflammatory cytokines, and up-regulation of MHCmolecules and co-stimulatory signals in antigen-presenting cells as wellas activating natural killer (NK) cells. The consequence of activationof the innate immune system mobilizes and amplifies specific adaptiveimmune responses involving both T- and B-cell effector functions.⁵⁷Thus, TLR stimuli serve to link innate and adaptive immunity⁵ byeliciting both primary and anamnestic immune responses.

TLR7 agonists stimulate virtually all subsets of lymphocytes withoutinducing dominant proinflammatory cytokine responses (unlike TLR4-5 or-8 agonists, which can be proinflammatory and therefore may exertsystemic toxicity).⁸ TLR7-active compounds therefore representcandidates as potential vaccine adjuvants and immune response modifiers.Dual TLR7 and TLR8 agonists also

In the 1970s and '80s a number of small molecules were synthesized andevaluated for antiviral activities owing to their pronounced Type Iinterferon (IFN-α and -β) inducing properties.¹²⁻¹⁶ The1H-imidazo[4,5-c]quinolines were found to be good Type I IFN inducers inhuman cell-derived assays.¹⁷ Imiquimod is FDA approved for the treatmentof basal cell carcinoma and actinic keratosis, and Gardiquimod is aanother imidazoquinoline TLR7 agonist.¹⁸ Several years later themechanistic basis of IFN induction by the imidazoquinolines was found tobe a consequence of TLR7 engagement and activation.¹⁹ Certainimidazoquinoline compounds have been approved for use as antiviralagents as well as immune modulating compounds. However, thestructure-activity relationship of the imidazoquinoline chemotype stillremains largely unexplored and new, useful imidazoquinoline basedcompounds are still being developed.

SUMMARY

Embodiments of the present disclosure include imidazoquinoline derivedcompounds of Formula I, as well as derivatives and analogues, andpharmaceutically acceptable salts of the compounds, where Formula I isrepresented by the following structure.

In embodiments of the imidazoquinoline derived compounds of the presentdisclosure of Formula I, R is selected from the group consisting of:—NH(R₅) and isothiocyanate; R₅ is selected from the group consisting ofhydrogen, acetyl, —CO-tert-Bu (-Boc), —CO—(CH₂)_(x)—R₆, C₁-C₁₆ alkyl,—CO-4-, —C(S)—NH—(CH₂)_(x)—NH—(CH₂)—NH—(CH₂)—NH₂,

a reporter moiety, a tissue-specific moiety, a peptide antigen moiety, aprotein antigen moiety, a polysaccharide antigen moiety, and a TLR2agonist moiety; R₆ is selected from the group consisting of hydrogen,alkyne, azido, carboxylic acid, and—CONH—(CH₂)_(x)—O—(CH₂)—O—(CH₂)_(x)—O—(CH₂)_(x)—R₇; R₇ is selected fromthe group consisting of amino, isothiocyanate, and—NH—CO—(CH₂)_(x)—CO₂H; R₈ is selected from a peptide antigen moiety or aprotein antigen moiety; and x is any integer from 1 to 10.

The present disclosure also includes imidazoquinoline derived compoundshaving the structure of Formula II, below, and derivatives andanalogues, and pharmaceutically acceptable salts of such compounds.

where, R₁ and R₃ are each independently selected from the groupconsisting of hydrogen, halogen, nitro, —NH₂, azido, hydroxyl, —CF₃,carboxylic acid, and —CO₂R₂; R₂ is a C₂-C₅ alkyl, and R₄ is selectedfrom the group consisting of: —NH(R₅) and isothiocyanate; R₅ is selectedfrom the group consisting of hydrogen, acetyl, —CO-tert-Bu (-Boc),—CO—(CH₂)_(x)—R₆, C₁-C₁₆ alkyl, —CO-4-,—C(S)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH₂,

a reporter moiety, a tissue-specific moiety, a peptide antigen moiety, aprotein antigen moiety, a polysaccharide antigen moiety, and a TLR2agonist moiety; R₆ is selected from the group consisting of hydrogen,alkyne (-, azido, carboxylic acid, and—CONH—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—R₇; R₇ is selectedfrom the group consisting of amino, isothiocyanate and—NH—CO—(CH₂)_(x)—CO₂H; R₈ is selected from a peptide antigen moiety or aprotein antigen moiety; and x is any integer from 1 to 10.

Embodiments of the present disclosure also include dendrimers and dimersof compounds of Formula I and Formula II as defined above, derivativesthereof, analogues thereof, and pharmaceutically acceptable saltsthereof. Dendrimers of the compounds of Formulas I and II include, butare not limited to, trimers and hexamers of compounds of Formula I andII, as well as derivatives, analogues, and pharmaceutically acceptablesalts of such dendrimers. The dimers of the present disclosure includedimers of compounds of Formulas I and II, as well as derivatives andanalogues, and pharmaceutically acceptable salts of such dimers.

The present disclosure also provides TLR7 agonists includingimidazoquinoline derived compounds chosen from compounds: 6c, 6d, 7c,7d, 8, (12), 13, 14, (15), (16), 17, (18), 19, (21), (23), 25, 26-28,30b, 31, 33, 35, 37, 39, 60, 61, 62, 64, and 65, as well as derivativesand analogues, and pharmaceutically acceptable salts of these compounds.

Embodiments of the present disclosure also include dual TLR7/TLR8agonists including imidazoquinoline derived compounds chosen fromcompounds 7c, 7d, (19), 39, (35), 41, and 52c, and derivatives andanalogues, and pharmaceutically acceptable salts of these compounds.

Embodiments of the present disclosure also include vaccine adjuvants andself-adjuvanting vaccines including the imidazoquinoline derivedcompounds of the present disclosure.

The present disclosure also includes methods of treatment of conditionssuch as, but not limited to, hepatitis, chronic myelogenous, and hairycell leukemia by administering to a host in need of treatment for thecondition an effective amount of an imidazoquinoline derived compound ofthe present disclosure coupled to a tissue-specific agent.

Embodiments of the present disclosure also include probes includingimidazoquinoline derived compounds of the present disclosure thatinclude a reporter moiety or are coupled to a reporter moiety that iscapable of producing a detectable signal. The present disclosure alsoincludes methods of imaging activation of TLR7 and/or TLR8 by contactinga sample including TLR7 and/or TLR8 with a probe of the presentdisclosure.

The present disclosure also includes TLR7 antagonists includingimidazoquinoline derived dimeric compounds of Formula III, andderivatives, analogues and pharmaceutically acceptable salts of thesecompounds, where Formula III is represented by the following structure:

wherein R₁ is selected from the group consisting of hydrogen, halogen,nitro, —NH₂, azido, hydroxyl, and —CF₃,R₃ is selected from the group consisting of hydrogen and —(CH₂)_(x)—NH₂,andx is any integer form 1 to 10.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be more readilyappreciated upon review of the detailed description of its variousembodiments, described below, when taken in conjunction with theaccompanying drawings.

FIG. 1 illustrates the structures of Imiquimod (1) and Gardiquimod (2).

FIGS. 2A and 2B are graphs of TLR-7 and TLR-8 agonistic activities ofcompounds 6a-b and 7c-d.

FIGS. 3A and 3B are graphs illustrating the TLR-7 and TLR-8 agonisticactivities of derivatives of compound 7d.

FIG. 4 is a graph of the activities of 7d, 26, 27, and 28 in reportergene assays using human TLR7.

FIG. 5 is a digital image of murine J774 cells treated with 10 nM of 28.An overlay of phase-contrast and epifluorescence images is depicted. Anexcitation filter at 562 nm and a long-pass emission filter (601-800)were used.

FIG. 6 illustrates the uptake of 26 in lymphocytic subsets as examinedby flow cytometry. Whole human blood was incubated with gradedconcentrations of 26 for 30 min, lymphocytes stained with cell surfacemarkers (anti-CD3-phycoerythrin [PE], and anti-CD56-PE-allophycocyanin).Erythrocytes were lysed, and 105 total events were acquired per sample.

FIGS. 7A and 7B are graphs showing induction of IFN-α, IFN-γ (FIG. 7A)and IL-12 and IL-18 (FIG. 7B) by 7d in human PBMCs. IFN and cytokinelevels were quantified by ELISA. Results of a representative experimentare shown.

FIG. 8 is a graph illustrating TLR7-agonistic activities ofimidazoquinoline analogues in a human TLR7-specific reporter gene assay.

FIG. 9 illustrates a deconvoluted positive-mode ESI-MS spectra of nativebovine α-lactalbumin (left) showing a mass of 14178.83 Da, andα-lactalbumin reacted with 5 eq. of 8, resulting in a stochasticcoupling of the adjuvant with the centroid of the mass distributioncorresponding to exactly 5 units of imidazoquinoline per proteinmolecule.

FIGS. 10A-10D are graphs illustrating immunoglobulin profiles in outbredCF-1 mice immunized on Day 0 with 50 μg/animal of α-lactalbumin, orα-lactalbumin covalently coupled with 5 equivalents of 8, orα-lactalbumin mixed with 5 equivalents of 7d. Animals (5 per cohort)were boosted once on Day 14 exactly as mentioned above, and bled on Day21. α-lactalbumin-specific immunoglobulin levels were quantified bystandard antibody-capture ELISA, performed in liquid handler-assisted384-well format.

FIG. 11 is a graph illustrating affinity IgG ELISA showing antibodytiter as a function of chaotrope (NaSCN) concentration. IgG titers onthe ordinate axis were calculated from absorbance values at 0.25 (whichcorresponds to 3a above that of naïve controls).

FIG. 12 illustrates covalent coupling of the thiol-specific maleimidederivative 21 with human serum albumin showing addition of a singleequivalent of 21 to albumin, as examined by LC-ESI-TOF. An excess (5equiv.) of 21 was used.

FIG. 13 is a graph of TLR7-agonistic activities ofimidazoquinoline-maltoheptaose conjugate in a human TLR7-specificreporter gene assay.

FIGS. 14A and 14B illustrate TLR-7 and TLR-8 agonistic activities ofcompounds 33, 35, 37 and 39.

FIGS. 15A and 15B are graphs illustrating TLR-7 and -8 agonisticactivity of ‘Click reaction’ derived imidazoquinoline dendrimer 43.

FIGS. 16A-16E illustrate proinflammatory cytokine induction in humanPBMCs. Note selective and complete loss of TLR8-associated cytokineinduction by the dendrimer 43 FIGS. 17A and 17B illustrate Type I andType II Interferon induction in human PBMCs. Note selective and completeloss of TLR8-associated IFN-γ by the dendrimer 43.

FIG. 18 illustrates ratios of Immune-1/pre-immune (after primaryvaccination) and Immune-2/pre-immune (after Boost-1) anti-α-lactalbuminIgG titers in rabbits FIGS. 19A and 19B are graphs illustrating TLR7 andTLR8 agonistic activities of the imidazoquinoline dimers in humanTLR-specific reporter gene assays.

FIGS. 20A and 20B are graphs of TLR7 (20A) and TLR8 (20B) antagonisticactivities of the imidazoquinoline dimers 47a-b and 49a-b in humanTLR-specific reporter gene assays.

FIG. 21 is a graph illustrating IFN-α induction by select dimers inhuman peripheral blood mononuclear cells. IFN-α was assayed by analytespecific ELISA after incubation of hPBMCs with graded concentrations ofthe test compound for 12h.

FIGS. 22A-22H illustrate inhibition of TLR7-mediated (FIGS. 22A-22D) andTLR8-mediated (FIGS. 22E-22H) proinflammatory cytokine production inhuman peripheral blood mononuclear cells by chloroquine or 47a.Proinflammatory cytokines were assayed by cytokine bead array methodsafter incubation of hPBMCs with graded concentrations of the testcompound for 12h in the presence of 10 μg/ml of either CL075 (TLR8agonist) or gardiquimod (TLR7 agonist).

FIGS. 23A-23D illustrate inhibition of TLR7-mediated (FIGS. 23A-23B) andTLR8-mediated (FIGS. 23C-3D) chemokine production in human peripheralblood mononuclear cells by chloroquine or 47a. Chemokines were assayedby cytokine bead array methods after incubation of hPBMCs with gradedconcentrations of the test compound for 12h in the presence of 10 μg/mlof either CL075 (TLR8 agonist) or gardiquimod (TLR7 agonist).

FIGS. 24A and 24B are graphs of the schild plot analyses of inhibitionof TLR7- (24A) and TLR8-induced (24B) activation. Experiments wereperformed in checker-board format, using a liquid handler, in 384-wellplates which permitted the concentrations of both agonist and antagonistto be varied simultaneously along the two axes of the plate. Eitherimidazoquinoline (TLR7-specific agonist) or CL075 (TLR8-specificagonist) was used at a starting concentration of 20 μg/mL, and weretwo-fold diluted serially (along the rows). Next, 47a or chloroquine wastwo-fold diluted serially in HEK detection medium (along columns).Reporter cells were then added, incubated, and NF-κB activation measuredas described in the text. A and A′ (Y-axis) are defined respectively asthe EC₅₀ value in the absence of antagonist, and the EC₅₀ values in thepresence of varying concentrations of antagonist.

FIGS. 25A and 25B are graphs illustrating TLR-2 (25A) and -7 (25B)agonistic activities of compounds 60-65.

FIGS. 26A-26E are graphs illustrating cytokine induction in hPBMCS bythe compounds 60-65.

FIG. 27 illustrates ratios of immune-1/pre-immune (after primaryvaccination) and Immune-2/pre-immune (after Boost-1) anti-α-lactalbuminIgG titers in rabbits using the hybrids as adjuvants. Also shown are theIMDQ (7d) and PAM₂CS, as well as IMDQ (7d)+PAM2CS mixture controls.

DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification that areincorporated by reference are incorporated herein by reference todisclose and describe the methods and/or materials in connection withwhich the publications are cited. The citation of any publication is forits disclosure prior to the filing date and should not be construed asan admission that the present disclosure is not entitled to antedatesuch publication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Unless otherwise indicated, any recited method can becarried out in the order of events recited or in any other order that islogically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, and the like, which are within theskill of the art. Such techniques are explained in the literature.

It must be noted that, as used in the specification and the appendedembodiments, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a support” includes a plurality of supports. Inthis specification and in the embodiments that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings unless a contrary intention is apparent.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise. In this disclosure, “consisting essentiallyof” or “consists essentially” or the like, when applied to methods andcompositions encompassed by the present disclosure refers tocompositions like those disclosed herein, but which may containadditional structural groups, composition components or method steps.Such additional structural groups, composition components or methodsteps, etc., however, do not materially affect the basic and novelcharacteristic(s) of the compositions or methods, compared to those ofthe corresponding compositions or methods disclosed herein. “Consistingessentially of” or “consists essentially” or the like, when applied tomethods and compositions encompassed by the present disclosure have themeaning ascribed in U.S. patent law, and the term is open-ended,allowing for the presence of more than that which is recited so long asbasic or novel characteristics of that which is recited is not changedby the presence of more than that which is recited, but excludes priorart embodiments.

Prior to describing the various embodiments, the following definitionsare provided and should be used unless otherwise indicated.

Definitions

In describing the disclosed subject matter, the following terminologywill be used in accordance with the definitions set forth below.

The following abbreviations are used in the present disclosure and havethe meanings ascribed below.

AP-1 activator protein-1APC allophycocyaninAPCs antigen presenting cellsCD cluster of differentiationCTL cytotoxic T lymphocytesDCs dendritic cellsDMAP 4-dimethylaminopyridine

DMF N,N-dimethylformamide

DMSO Dimethyl sulfoxideEC₅₀ half-maximal effective concentrationEDCl.HCl 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochlorideELISA enzyme linked immunosorbent assayESI-TOF electrospray ionization-time of flight

FDA Food and Drug Administration

FITC fluorescein isothiocyanateFSC forward scatterHATU 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphateHBTU 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphateHEK-293 human embryonic kidney 293HSA human serum albuminIFN interferonIg immunoglobulinIL interleukinLPS lipopolysaccharidem-CPBA meta-chloroperoxy benzoic acidMHC major histocompatibility complexN-Boc N-tert-butyl carbamateNF-κB nuclear factor-kappa BNK cells natural killer cellsPAMP pathogen associated molecular patternPBMCs peripheral blood mononuclear cellsPE phycoerythrinRNA ribonucleic acidsAP secreted alkaline phosphataseSAR structure activity relationshipS_(N)Ar aromatic nucleophilic substitutionSSC side scatterssRNA single stranded RNATh1 helper T-type 1THF tetrahydrofuranTLRs toll like receptors

The term “nucleic acid” as used herein refers to any natural andsynthetic linear and sequential arrays of nucleotides and nucleosides,for example cDNA, genomic DNA, mRNA, tRNA, oligonucleotides,oligonucleosides and derivatives thereof. For ease of discussion, suchnucleic acids may be collectively referred to herein as “constructs,”“plasmids,” or “vectors.” Representative examples of the nucleic acidsof the present disclosure include bacterial plasmid vectors includingexpression, cloning, cosmid and transformation vectors such as, but notlimited to, pBR322, animal viral vectors such as, but not limited to,modified adenovirus, influenza virus, polio virus, pox virus,retrovirus, insect viruses (baculovirus), and the like, vectors derivedfrom bacteriophage nucleic acid, and synthetic oligonucleotides likechemically synthesized DNA or RNA. The term “nucleic acid” furtherincludes modified or derivatized nucleotides and nucleosides such as,but not limited to, halogenated nucleotides such as, but not only,5-bromouracil, and derivatized nucleotides such as biotin-labelednucleotides.

The term “isolated nucleic acid” as used herein refers to a nucleic acidwith a structure (a) not identical to that of any naturally occurringnucleic acid or (b) not identical to that of any fragment of a naturallyoccurring genomic nucleic acid spanning more than three separate genes,and includes DNA, RNA, or derivatives or variants thereof. The termcovers, for example, (a) a DNA which has the sequence of part of anaturally occurring genomic molecule but is not flanked by at least oneof the coding sequences that flank that part of the molecule in thegenome of the species in which it naturally occurs; (b) a nucleic acidincorporated into a vector or into the genomic nucleic acid of aprokaryote or eukaryote in a manner such that the resulting molecule isnot identical to any vector or naturally occurring genomic DNA; (c) aseparate molecule such as a cDNA, a genomic fragment, a fragmentproduced by polymerase chain reaction (PCR), ligase chain reaction (LCR)or chemical synthesis, or a restriction fragment; (d) a recombinantnucleotide sequence that is part of a hybrid gene, e.g., a gene encodinga fusion protein, and (e) a recombinant nucleotide sequence that is partof a hybrid sequence that is not naturally occurring. Isolated nucleicacid molecules of the present disclosure can include, for example,natural allelic variants as well as nucleic acid molecules modified bynucleotide deletions, insertions, inversions, or substitutions.

It is advantageous for some purposes that a nucleotide sequence is inpurified form. The term “purified” in reference to nucleic acidrepresents that the sequence has increased purity relative to thenatural environment.

The terms “polynucleotide,” “oligonucleotide,” and “nucleic acidsequence” are used interchangeably herein and include, but are notlimited to, coding sequences (polynucleotide(s) or nucleic acidsequence(s) which are transcribed and translated into polypeptide invitro or in vivo when placed under the control of appropriate regulatoryor control sequences); control sequences (e.g., translational start andstop codons, promoter sequences, ribosome binding sites, polyadenylationsignals, transcription factor binding sites, transcription terminationsequences, upstream and downstream regulatory domains, enhancers,silencers, and the like); and regulatory sequences (DNA sequences towhich a transcription factor(s) binds and alters the activity of agene's promoter either positively (induction) or negatively(repression)). No limitation as to length or to synthetic origin issuggested by the terms described herein.

The term “peptide” or “polypeptide” as used herein refers to proteinsand fragments thereof. Peptides are disclosed herein as amino acidresidue sequences. Those sequences are written left to right in thedirection from the amino to the carboxy terminus. In accordance withstandard nomenclature, amino acid residue sequences are denominated byeither a three letter or a single letter code as indicated as follows:Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid(Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E),Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu,L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F),Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp,W), Tyrosine (Tyr, Y), and Valine (Val, V).

The term “variant” refers to a peptide or polynucleotide that differsfrom a reference peptide or polynucleotide, but retains essentialproperties. A typical variant of a peptide differs in amino acidsequence from another, reference peptide. Generally, differences arelimited so that the sequences of the reference peptide and the variantare closely similar overall and, in many regions, identical. A variantand reference peptide may differ in amino acid sequence by one or moremodifications (e.g., substitutions, additions, and/or deletions). Avariant of a peptide includes conservatively modified variants (e.g.,conservative variant of about 75, about 80, about 85, about 90, about95, about 98, about 99% of the original sequence). A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a peptide may be naturally occurring, such as anallelic variant, or it may be a variant that is not known to occurnaturally.

The present disclosure includes peptides which are derivable from thenaturally occurring sequence of the peptide. A peptide is said to be“derivable from a naturally occurring amino acid sequence” if it can beobtained by fragmenting a naturally occurring sequence, or if it can besynthesized based upon knowledge of the sequence of the naturallyoccurring amino acid sequence or of the genetic material (DNA or RNA)that encodes this sequence. Included within the scope of the presentdisclosure are those molecules which are said to be “derivatives” of apeptide. Such a “derivative” or “variant” shares substantial similaritywith the peptide or a similarly sized fragment of the peptide and iscapable of functioning with the same biological activity as the peptide.

A derivative of a peptide is said to share “substantial similarity” withthe peptide if the amino acid sequences of the derivative is at least80%, at least 90%, at least 95%, or the same as that of either thepeptide or a fragment of the peptide having the same number of aminoacid residues as the derivative.

The protein or peptide derivatives of the present disclosure includefragments which, in addition to containing a sequence that issubstantially similar to that of a naturally occurring peptide maycontain one or more additional amino acids at their amino and/or theircarboxy termini. Similarly, the invention includes peptide fragmentswhich, although containing a sequence that is substantially similar tothat of a naturally occurring peptide, may lack one or more additionalamino acids at their amino and/or their carboxy termini that arenaturally found on the peptide.

The disclosure also encompasses the obvious or trivial variants of theabove-described fragments which have inconsequential amino acidsubstitutions (and thus have amino acid sequences which differ from thatof the natural sequence) provided that such variants have an activitywhich is substantially identical to that of the above-describedderivatives. Examples of obvious or trivial substitutions include thesubstitution of one basic residue for another (i.e. Arg for Lys), thesubstitution of one hydrophobic residue for another (i.e. Leu for lie),or the substitution of one aromatic residue for another (i.e. Phe forTyr), etc.

Modifications and changes can be made in the structure of the peptidesof this disclosure and still obtain a molecule having similarcharacteristics as the peptide (e.g., a conservative amino acidsubstitution). For example, certain amino acids can be substituted forother amino acids in a sequence without appreciable loss of activity.Because it is the interactive capacity and nature of a peptide thatdefines that peptide's biological functional activity, certain aminoacid sequence substitutions can be made in a peptide sequence andnevertheless obtain a peptide with like properties.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a peptide is generallyunderstood in the art. It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still result in a peptide with similar biological activity.Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics. Those indices are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant peptide, which inturn defines the interaction of the peptide with other molecules, suchas enzymes, substrates, receptors, antibodies, antigens, and the like.It is known in the art that an amino acid can be substituted by anotheramino acid having a similar hydropathic index and still obtain afunctionally equivalent peptide. In such changes, the substitution ofamino acids whose hydropathic indices are within ±2 is preferred, thosewithin +1 are particularly preferred, and those within ±0.5 are evenmore particularly preferred.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly, where the biological functional equivalentpeptide or peptide thereby created is intended for use in immunologicalembodiments. The following hydrophilicity values have been assigned toamino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); proline (−0.5±1); threonine (−0.4); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine(−2.5); tryptophan (−3.4). It is understood that an amino acid can besubstituted for another having a similar hydrophilicity value and stillobtain a biologically equivalent, and in particular, an immunologicallyequivalent peptide. In such changes, the substitution of amino acidswhose hydrophilicity values are within ±2 is preferred, those within ±1are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include (original residue: exemplary substitution): (Ala:Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gln:Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (Ile: Leu, Val), (Leu:lie, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip:Tyr), (Tyr: Trp, Phe), and (Val: lie, Leu).

The term “fragment” as used herein to refer to a nucleic acid (e.g.,cDNA) refers to an isolated portion of the subject nucleic acidconstructed artificially (e.g., by chemical synthesis) or by cleaving anatural product into multiple pieces, using restriction endonucleases ormechanical shearing, or a portion of a nucleic acid synthesized by PCR,DNA polymerase or any other polymerizing technique well known in theart, or expressed in a host cell by recombinant nucleic acid technologywell known to one of skill in the art. The term “fragment” as usedherein may also refer to an isolated portion of a polypeptide, whereinthe portion of the polypeptide is cleaved from a naturally occurringpolypeptide by proteolytic cleavage by at least one protease, or is aportion of the naturally occurring polypeptide synthesized by chemicalmethods well known to one of skill in the art.

As used herein, the term “moiety” means a chemical group on a compoundor capable of being coupled to a compound that includes a functionalgroup/subunit. As used herein, a “moiety” may include a compound with aspecific function that is a part of a larger compound or capable ofbeing coupled to a different compound to form a larger compound. Forinstance, a “reporter moiety” is a chemical group that includes areporter compound (e.g., a fluorescent dye molecule) that is coupled toor adapted to be coupled to another compound.

As used herein, the term “agonist” indicates a compound that induces areceptor molecule, for instance, a ligand that binds with and activatesa receptor molecule. In embodiments of the present disclosure,imidazoquinoline derived compounds of the present disclosure are ligandsthat can activate certain receptors in a host immune system, such as TLR7 and TLR8, thereby inducing the receptors to generate an immunologicalresponse. Thus, in embodiments, the imidazoquinoline derived compoundsof the present disclosure can be TLR7 or dual TLR7/TLR8 agonists.

The term “reporter molecule” for use in the present disclosure includesany substance or group capable of being coupled to the imidazoquinolinederived compounds of the present disclosure (e.g., attached/bound to theimidazoquinoline derived compounds) and capable of producing adetectable signal, such as, but not limited to, molecules withparticular optical, electrical, acoustic and magnetic properties thatcan generate a distinguishable signals different from the detectingtarget, such as, for instance, fluorescent molecules, fluorescent dyes,fluorescent quantum dots, MRI agents, and the like. Such reportermolecules may have the inherent ability to produce a detectable signal,or may produce a detectable signal in the presence of an activator. Inembodiments, the reporter molecule produces a signal that isdistinguishable from background signals. In embodiments the reportermolecule is “covalently coupled” to the imidazoquinoline derivedcompounds, meaning it is attached to the imidazoquinoline derivedcompound by a covalent bond. As used herein, a “reporter moiety”includes a reporter molecule as described above that is coupled to orcapable of being coupled to imidazoquinoline derived compounds of thepresent disclosure.

In some exemplary embodiments, a fluorophore or a fluorescent dye isused as the reporter molecule to label the host and reference samples.Suitable dye molecules include, but are not limited to, Alexa 350, Alexa430, Alexa 488, Alexa 532, Alexa 546, Alexa 568, and Alexa 594 dyes,AMCA, LuciferYellow, fluorescein, luciferins, aequorins, rhodamine 6G,tetramethylrhodamine or Cy3, Cy5, lissamine rhodamine B, amine-bearingfluorophores, such as the bora-diazaindacene dye, BODIPY-TR-cadaverine,and Texas Red, (the numbers in the Alexa names indicate the approximateexcitation wavelength maximum in nm).

A “probe” according to the present disclosure, refers to a compound usedfor detecting a target, such as by binding or otherwise interacting witha target in such a way that interaction between the probe and the targetcan be detected. Probes may be used to detect a target either in vivo(e.g, in host, living cell, or tissue sample) or in vitro (e.g., in asample, culture, composition). In embodiments a probe may include aportion that interacts with the target (e.g., by binding the target) andanother portion that produces a detectable signal to allow detection(e.g., by imaging) of the target/probe complex. In embodiments of thepresent disclosure, a probe may include an imidazoquinoline derivedcompound of the present disclosure coupled to a reporter molecule.

The terms “treat”, “treating”, and “treatment” are an approach forobtaining beneficial or desired clinical results. Specifically,beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease,stabilization (e.g., not worsening) of disease, delaying or slowing ofdisease progression, substantially preventing spread of disease,amelioration or palliation of the disease state, and remission (partialor total) whether detectable or undetectable. In addition, “treat”,“treating”, and “treatment” can also mean prolonging survival ascompared to expected survival if not receiving treatment and/or can betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. As used herein, the terms“prophylactically treat” or “prophylactically treating” referscompletely, substantially, or partially preventing a disease/conditionor one or more symptoms thereof in a host. Similarly, “delaying theonset of a condition” can also be included in “prophylacticallytreating”, and refers to the act of increasing the time before theactual onset of a condition in a patient that is predisposed to thecondition.

The term “host” or “organism” as used herein includes humans, mammals(e.g., cats, dogs, horses, etc.), insects, living cells, and otherliving organisms. A living organism can be as simple as, for example, asingle eukaryotic cell or as complex as a mammal. Typical hosts to whichembodiments of the present disclosure relate will be insects (e.g.,Drosophila melanogaster) mammals, particularly primates, especiallyhumans. For veterinary applications, a wide variety of subjects will besuitable, e.g., livestock such as cattle, sheep, goats, cows, swine, andthe like; poultry such as chickens, ducks, geese, turkeys, and the like;and domesticated animals particularly pets such as dogs and cats. Forsome applications, hosts may also include plants. For diagnostic orresearch applications, a wide variety of mammals will be suitablesubjects, including rodents (e.g., mice, rats, hamsters), rabbits,primates, and swine such as inbred pigs and the like. Additionally, forin vitro applications, such as in vitro diagnostic and researchapplications, body fluids and cell samples of the above subjects will besuitable for use, such as mammalian (particularly primate such as human)blood, urine, or tissue samples, or blood, urine, or tissue samples ofthe animals mentioned for veterinary applications. Hosts that are“predisposed to” condition(s) can be defined as hosts that do notexhibit overt symptoms of one or more of these conditions but that aregenetically, physiologically, or otherwise at risk of developing one ormore of these conditions.

By “administration” is meant introducing a compound of the presentdisclosure into a subject; it may also refer to the act of providing acomposition of the present disclosure to a subject (e.g., byprescribing). The term “therapeutically effective amount” as used hereinrefers to that amount of the compound being administered which willrelieve or prevent to some extent one or more of the symptoms of thecondition to be treated. In reference to conditions/diseases that can bedirectly treated with a composition of the disclosure, a therapeuticallyeffective amount refers to that amount which has the effect ofpreventing the condition/disease from occurring in an animal that may bepredisposed to the disease but does not yet experience or exhibitsymptoms of the condition/disease (prophylactic treatment), alleviationof symptoms of the condition/disease, diminishment of extent of thecondition/disease, stabilization (e.g., not worsening) of thecondition/disease, preventing the spread of condition/disease, delayingor slowing of the condition/disease progression, amelioration orpalliation of the condition/disease state, and combinations thereof. Theterm “effective amount” refers to that amount of the compound beingadministered which will produce a reaction that is distinct from areaction that would occur in the absence of the compound. In referenceto embodiments of the disclosure including the imidazoquinoline derivedcompounds of the disclosure as adjuvants or self-adjuvanting vaccines,an “effective amount” is that amount which increases the immunologicalresponse in the recipient over the response that would be expectedwithout administration of the compound. In reference to probes, an“effective amount” or “detectably effective amount” would be that amountwhich produces a detectable signal that is distinguishable frombackground signal.

Compositions and immunogenic preparations of the present disclosure,including vaccine compositions, (including the imidazoquinoline derivedcompounds of the present disclosure, with or without an additionalantigen) and capable of inducing protective immunity in a suitablytreated host and a suitable carrier therefor are provided. “Immunogeniccompositions” are those which result in specific antibody production orin cellular immunity when injected into a host. Such immunogeniccompositions or vaccines are useful, for example, in immunizing hostsagainst infection and/or damage caused by viruses and/or bacteria.

By “immunogenic amount” or “immunogenic effective amount” is meant anamount capable of eliciting the production of antibodies directedagainst the virus and/or bacteria, in the host to which the vaccine hasbeen administered.

As used herein, the term “adjuvant” indicates a compound that inducesand/or enhances an immunological response in a host. The adjuvants ofthe present disclosure induce immunological responses by activatingtoll-like receptor (TLR) 7 or by activating TLR 7 and TLR8. Some of theadjuvants of the present disclosure may also induce other immunologicalresponses in the host in addition to the activation of TLR 7 and/or 8,such as by stimulating interferons (IFN). In general, an “immunologicalresponse” refers to a response by the host's immune system to a stimuli,in this case, and adjuvant. Adjuvants that “enhance” an immunologicalresponse in a host induce a stronger immunological response to anantigen or other immunological stimulus in the host than would be seenby the administration of an antigen and/or stimulus alone.

The term “self-adjuvant” or “self-adjuvanting vaccine” indicates acompound and/or vaccine (e.g., an antigen that induces an immuneresponse) where the adjuvant effect is induced by the compound itselfwithout the need for a separate adjuvant compound. In embodiments of aself-adjuvant of the present disclosure, an adjuvant molecule iscovalently coupled to an antigen. This is in contrast to an antigen andan adjuvant molecule that are physically separate from each other (e.g.,not coupled), even though they may be co-administered.

As used herein, the term “tissue-specific” or “tissue-specific moiety”or “tissue-specific agent” refers to compounds or moieties of compoundsthat have a specific affinity for a certain tissue and/or location in ahost system. As such, “tissue-specific” agents can be used to direct acompound to a desired tissue or location in a host sample or system. Inembodiments of the present disclosure, a tissue-specific moietyincluding a tissue-specific compound can be coupled to animidazoquinoline derived compound of the present disclosure to assist indirecting and locating the imidazoquinoline derived compound to adesired tissue and/or location in a host sample or system.

As used herein in reference to the imidazoquinoline derived compounds ofthe present disclosure, the term “derivative” refers to a new chemicalentity that is derived from A (Gardiquimod, in the example illustratedimmediately below) by means of chemical transformation. Derivatives aredistinguished from “analogues” of the compounds of the presentdisclosure, which refers to a new chemical entity that is structurallyrelated to A, but which cannot be obtained from A, but has to besynthesized using other precursors. Below, the top row representsGardiquimod and two of its derivatives, and the bottom row depictsGardiquimod and two of its analogues.

The term “imidazoquinoline derived compounds” refers to theimidazoquinoline derivatives and imidazoquinoline analogues of thepresent disclosure, as well as intermediates of such compounds.

“Dendrimers”, as used herein, refers to branched compounds made of twoor more, typically identical, sub-units or moieties. Typically, and asused in the present disclosure, the sub-units are covalently bound toeach other. In the compounds of the present disclosure, dendrimersinclude two more identical imidazoquinoline derived compounds of thepresent disclosure that are covalently linked. In embodiments,dendrimers of the present disclosure include, but are not limited to,“dimmers” (two sub-units), “trimers” (three sub-units), and “hexamers”(six sub-units) of the imidazoquinoline derived compounds of the presentdisclosure.

“Pharmaceutically acceptable salt” refers to those salts which retainthe biological effectiveness and properties of the free bases and whichare obtained by reaction with inorganic or organic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid,succinic acid, tartaric acid, citric acid, and the like.

A “pharmaceutical composition” refers to a mixture of one or more of thecompounds described herein, derivatives thereof, or pharmaceuticallyacceptable salts thereof, with other chemical components, such aspharmaceutically acceptable carriers and excipients. One purpose of apharmaceutical composition is to facilitate administration of a compoundto the organism.

As used herein, a “pharmaceutically acceptable carrier” refers to acarrier or diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound.

As used herein, “alkyl” or “alkyl group” refers to a saturated aliphatichydrocarbon radical which can be straight or branched, having 1 to 20carbon atoms or the stated range of carbon atoms, where the stated rangeof carbon atoms includes each intervening integer individually, as wellas sub-ranges. Examples of alkyl include, but are not limited to methyl,ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, ands-pentyl.

As used herein, “alkenyl” or “alkenyl group” refers to an aliphatichydrocarbon radical which can be straight or branched, containing atleast one carbon-carbon double bond, having 2 to 20 carbon atoms or thestated range of carbon atoms, where the stated range of carbon atomsincludes each intervening integer individually, as well as sub-ranges.Examples of alkenyl groups include, but are not limited to, ethenyl,propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl,heptenyl, octenyl, decenyl, and the like.

DISCUSSION

Embodiments of the present disclosure include novel imidazoquinolinederived compounds, including derivatives and analogues of imiquimod andgardiquimod. In embodiments, the imidazoquinoline derived compounds arecapable of activating TLR7 or dual activation of TLR7/TLR8. Embodimentsof the present disclosure also include compositions including theimidazoquinoline derived compounds, methods of using theimidazoquinoline derived compounds, and methods of synthesis of theimidazoquinoline derived compounds. The imidazoquinoline derivedcompounds may be useful as adjuvants, probes, immunogenic compositions,and/or therapeutic compositions. The imidazoquinoline derived compoundsmay be coupled to reporter molecules, antigens, and/or other moleculesfor use as probes, vaccine adjuvants, and/or self-adjuvanting vaccines.Further embodiments of the disclosure include dendrimers (e.g., dimmers,trimers, hexamers, etc.) of the imidazoquinoline derived compounds, anduse of the dendrimers for activation of LTR7 or TLR7/TLR8. Embodimentsof the present disclosure also include dual TLR2/TLR7 hybrid agoniststhat include imidazoquinoline derived compounds of the presentdisclosure conjugated to PMM2CS TLR2 agonists. In embodiments, some ofthe imidazoquinoline derived compounds of the present disclosure may beused to treat certain disease conditions. These and other embodiments ofthe present disclosure will be described in greater detail below.

Novel Imidazoquinoline Derived Compounds

Although the imidazoquinoline compounds Imiquimod and Gardiquimod andmany of their derivatives are known, the present disclosure providesnovel derivatives and analogues of these compounds. Embodiments of thepresent disclosure include imidazoquinoline derived compounds having thefollowing formula, as well as derivatives, analogues, andpharmaceutically acceptable salts of such compounds.

In embodiments of the imidazoquinoline derived compounds of the presentdisclosure of Formula I,

R is selected from the group consisting of: —NH(R₅) and isothiocyanate(—NCS);

R₅ is selected from the group consisting of hydrogen (—H), acetyl (e.g.,a group that includes —COCH₃), —CO-tert-Bu (-Boc), —CO—(CH₂)_(x)—R₆,C₁-C₁₆ alkyl, —CO-4-(phenylboronic acid),—C(S)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH₂,

a reporter moiety, a tissue-specific moiety, a peptide antigen moiety, aprotein antigen moiety, a polysaccharide antigen moiety, and a TLR2agonist moiety;

R₆ is selected from the group consisting of hydrogen (—H), alkyne (e.g.,a group that includes a carbon-carbon triple bond such as —C≡CH), azido(e.g., a group that includes a —N₃), carboxylic acid (e.g., a group thatincludes a —CO₂H), and—CONH—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—R₇;

R₇ is selected from the group consisting of amino (e.g., a group thatincludes a —NH₂), isothiocyanate (e.g., a group that includes a —NCS)and —NH—CO—(CH₂)_(x)—CO₂H;

R₈ is selected from a peptide antigen moiety or a protein antigenmoiety; and

x is any integer from 1 to 10.

In embodiments of the imidazoquinoline derived compounds of Formula I ofthe present disclosure where R is a reporter moiety, the reporter moietyincludes a reporter molecule, such as, but not limited to, a fluorescentmolecule or an MRI agent. In embodiments, the reporter molecule isselected from fluorescein, rhodamine B, bora-diazaindacene dye, andBODIPY-TR-cadaverine, and biotin. In embodiments the reporter moietyincludes an isothiocyanate modified reporter molecule.

In embodiments of the imidazoquinoline derived compounds of Formula I ofthe present disclosure where R is a tissue-specific moiety, thetissue-specific moiety includes a tissue specific molecule. Inembodiments, the tissue-specific molecule includes tissue-specificagents such as, but not limited to, galactosyl and vitamins such asfolic acid, biotin, and pyridoxal.

In embodiments of the imidazoquinoline derived compounds of Formula I ofthe present disclosure where R is a peptide or protein antigen moiety ora polysaccharide antigen moiety, the moiety includes a peptide orprotein antigen or a polysaccharide antigen, respectively. Inembodiments, the protein antigen is α-lactalbumin. In embodiments wherethe antigen moiety is a peptide antigen, the peptide antigen can be, butis not limited to, tri-glycine methyl ester model peptide, andglutathione peptide. In embodiments, the polysaccharide antigen ismaltoheptaose.

In embodiments of the imidazoquinoline derived compounds of Formula I ofthe present disclosure where R is a TLR2 agonist moiety, the TLR2agonist moiety includes a TLR2 agonist, such as, but not limited to,S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cysteinyl-S-serine (PAM(2)CS)and derivatives of PAM(2)CS.

Embodiments of the present disclosure also include dimers and dendrimersof compounds of Formula I, including, but not limited to, dimers,trimers, and hexamers of compounds of Formula I, as well as derivatives,analogues, and pharmaceutically acceptable salts of such dendrimers.

Additional details regarding the compounds and methods of makingcompounds of Formula I, its precursors and derivatives can be found inExamples 1 and 2 below, as well as the other Examples. Embodiments ofcompounds of Formula I include, but are not limited to, compounds 6d,7d, 8, 12, 13, 14, 15, 16, 17, 18, 19, 21, 23, 25, 26, 27, 28, 29, 30a,30b, 31, 33, 35, 37, 39, 60, 61, 62, 63, 64, and 65, and derivatives andanalogues of these compounds or pharmaceutically acceptable saltsthereof, which are described in detail in the Examples below.

In embodiments of the imidazoquinoline derived compounds of the presentdisclosure of Formula I, R is selected from the group consisting of:—NH(R₅) and isothiocyanate (—NCS), where R₅ is selected from the groupconsisting of hydrogen (—H), acetyl (e.g., a group that includes—COCH₃), —CO-tert-Bu (-Boc), —CO—(CH₂)_(x)—R₆, C₁-C₁₆ alkyl,—CO-4-(phenylboronic acid),—C(S)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH₂,

R₆ is selected from the group consisting of hydrogen (—H), alkyne (e.g.,a group that includes —C≡CH), azido (—N₃), carboxylic acid (e.g., agroup that includes —CO₂H), and—CONH—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—R₇; R₇ is selectedfrom the group consisting of amino (e.g., a group including —NH₂),isothiocyanate (e.g., a group including —NCS) and —NH—CO—(CH₂)_(x)—CO₂H;R₈ is selected from a peptide antigen moiety or a protein antigenmoiety, and x is any integer from 1 to 10. In such embodiments, theimidazoquinoline derived compounds of the present disclosure of FormulaI can also include a moiety coupled to the R group, where the moiety isselected from a reporter moiety, a tissue-specific moiety, a peptideantigen moiety, a protein antigen moiety, a polysaccharide antigenmoiety, and a TLR2 agonist moiety, as described in greater detail below.

The present disclosure also includes imidazoquinoline derived compoundshaving the structure of Formula II, below, and derivatives, analogues,and pharmaceutically acceptable salts of such compounds. Thesubstituents shown on the N1-benzyl unit can be independently at theortho, meta, or para positions, and R₁-R₄ are as defined below.

Where,

R₁ and R₃ are each independently selected from the group consisting ofhydrogen, halogen (e.g., a group that includes —Cl, —Br, —F), nitro(e.g., a group that includes —NO₂), —NH₂, azido (e.g., a group thatincludes —N₃), hydroxyl (e.g., a group that includes —OH), —CF₃,carboxylic acid (e.g., a group that includes —CO₂H) and —CO₂R₂;

R₂ is a C₂-C₅ alkyl, and

R₄ selected from the group consisting of: —NH(R₅) and isothiocyanate(e.g., a group that includes —NCS);

R₅ is selected from the group consisting of hydrogen (—H), acetyl (e.g.,a group that includes —COCH₃), —CO-tert-Bu (-Boc), —CO—(CH₂)_(x)—R₆,C₁-C₁₆ alkyl, —CO-4-(phenylboronic acid),—C(S)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH₂,

a reporter moiety, a tissue-specific moiety, a peptide antigen moiety, aprotein antigen moiety, a polysaccharide antigen moiety, and a TLR2agonist moiety;

R₆ is selected from the group consisting of hydrogen (—H), alkyne (e.g.,a group that includes a carbon-carbon triple bond such as —C≡CH), azido(e.g., a group including —N₃), carboxylic acid (e.g., a group thatincludes a —CO₂H), and—CONH—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—R₇;

R₇ is selected from the group consisting of amino (e.g., a group thatincludes a —NH₂), isothiocyanate (e.g., a group that includes a —NCS)and —NH—CO—(CH₂)_(x)—CO₂H;

R₈ is selected from a peptide antigen moiety or a protein antigenmoiety; and

x is any integer from 1 to 10.

In embodiments of the imidazoquinoline derived compounds of Formula IIof the present disclosure R₄ is any of the substituents described abovefor R of Formula I. In embodiments of the imidazoquinoline derivedcompounds of the present disclosure of Formula II, R₄ is selected fromthe group consisting of: —NH(R₅) and isothiocyanate (e.g., a group thatincludes —NCS), where R₅ is selected from the group consisting ofhydrogen (—H), acetyl (e.g., a group that includes —COCH₃), —CO-tert-Bu(-Boc), —CO—(CH₂)_(x)—R₆, C₁-C₁₆ alkyl, —CO-4-(phenylboronic acid),—C(S)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH₂,

R₆ is selected from the group consisting of hydrogen (—H), alkyne (e.g.,a group that includes —C≡CH), azido (—N₃), carboxylic acid (e.g., agroup that includes —CO₂H), and—CONH—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—R₇; R₇ is selectedfrom the group consisting of amino (e.g., a group including —NH₂),isothiocyanate (e.g., a group including —NCS) and —NH—CO—(CH₂)_(x)—CO₂H;R₈ is selected from a peptide antigen moiety or a protein antigenmoiety, and x is any integer from 1 to 10. In such embodiments, theimidazoquinoline derived compounds of the present disclosure of FormulaII can also include a moiety coupled to the R₄ group, where the moietyis selected from a reporter moiety, a tissue-specific moiety, a peptideantigen moiety, a protein antigen moiety, a polysaccharide antigenmoiety, and a TLR2 agonist moiety, as described in greater detail below.

Some examples of compounds of the present disclosure having Formula IIinclude, but are not limited to: 6c, and 7c, and derivatives andanalogues thereof or pharmaceutically acceptable salts thereof, as wellas the compounds of Formula I set forth above.

Many of the imidazoquinoline derived compounds of Formula I and II aboveand their various derivatives, and analogues thereof are described ingreater detail in the discussion and examples below.

Embodiments of the present disclosure also include derivatives andanalogues of the compounds of Formula I and Formula II above, including,but not limited to, dendrimers and dimers of the compounds of Formula Iand Formula II. These and other dendrimers and dimers will be discussedin greater detail below.

Structure-Activity Relationships in Human TLR 7-Active and TLR7/8 DualActive Imidazoquinoline Analogues

Toll-like receptors (TLR)-7/-8 are innate immune receptors present inthe endosomal compartment that are activated by single-stranded RNA(ssRNA) molecules of viral as well as nonviral origin, inducing theproduction of inflammatory cytokines necessary for the development ofadaptive immunity. Molecules that induce (TLR)-7/-8 (e.g., agonists)represent potential vaccine adjuvants. Synthetic small molecule agonistsof TLR7 include the imidazoquinoline class of compounds such asGardiquimod[1-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol].With the goal of developing more potent TLR7 agonists as adjuvants,various derivatives and analogues of gardiquimod were synthesized and adetailed SAR study on the imidazoquinoline chemotype was performed,which led to the discovery of highly potent, lipophilic, human TLR7agonists. Details regarding the imidazoquinoline derived compounds withTLR7 and/or TLR7/8 agonistic activity, methods of synthesizing theimidazoquinoline derived compounds, and methods of using such compoundsare described in greater detail in the Examples below.

The present disclosure provides adjuvants including imidazoquinolinederived compounds chosen from compounds of Formulas I and II andderivatives and analogues thereof and pharmaceutically acceptable saltsthereof, where the compound is capable of activating TLR7. Inembodiments of the present disclosure, imidazoquinoline derivedcompounds include compounds of Formula I and/or Formula II, including,but not limited to, compound 7d and derivatives and analogues ofcompound 7d. In embodiments, these imidazoquinoline derived compoundshave been demonstrated to activate TLR7, as discussed in more detail inthe Examples. In embodiments some of the compounds capable of inducingTLR7 activity include, but are not limited to, compounds 6c, 6d, 7c, 7d,8, 12, 13, 14, 15), 16, 17, 18, 19, 21, 23, 25, 26-28, 30b, 31, 33, 35,37, 39, 41, 43, 51a-d, 52a-c, 55a-c, 60, 61, 62, 64, 65, andderivatives, analogs, and pharmaceutically acceptable salts of thosecompounds.

In some embodiments, the imidazoquinoline derived compounds of thepresent disclosure are capable of dual activation of both TLR7 and TLR8.While traditionally it had been thought that activation of TLR8 might beundesirable due to potential systemic proinflammatory responses, it hasnow been discovered that in some instances dual activation of both TLR7and TLR8 provides advantages and is desirable. It has been found that insome cases, induction of immunological responses from TLR7 alone may notproduce as much reactivity as dual induction, and in such cases, dualinduction of TLR7/8 provides additional immunological response. Forinstance, in the case of infants and very young babies, the induction ofTLR aloe may not produce a sufficient response to generate an effectiveimmune response. However, in some embodiments, dual induction of TLR7and TLR8 together can produce an effective response. The dual inductionalso induces the production of IL12 and IL18, which also plays a role ininducing immunity. In embodiments, some of the compounds capable ofinducing both TLR7 and TLR8 activity include imidazoquinoline derivedcompounds 7c, 7d, 19 and derivatives or analogs of those compounds.

Embodiments of the present disclosure also include TLR7 agonistsincluding, but not limited to, the following imidazoquinoline derivedcompounds: 6c, 6d, 7c, 7d, 8, 12, 13, 14, 15, 16, 17, 18, 19, 21, 23,25, 26-28, 30b, 31, 33, 35, 37, 39, 41, 43, 51a-d, 52a-c, 55a-c, 60, 61,62, 64, 65, and derivatives and/or analogs of those compounds. Thepresent disclosure also provides compositions including a TLR7 agonistor a dual TLR7/8 agonist of the present disclosure. In embodiments, theTLR 7/8 agonists include, but are not limited to, compounds 7c, 7d, 19,39, 35, 41, 52c, or derivatives of each of those or analogues thereof.

Further embodiments of the present disclosure include vaccine adjuvantsincluding the imidazoquinoline derived compounds of the presentdisclosure. In representative embodiments, the imidazoquinoline derivedcompounds that can be used as vaccine adjuvants include, but are notlimited to, the compounds listed above as representative TLR7 and/orTLR7/TLR8 agonists and derivatives or analogues of those compounds.

In other embodiments, the present disclosure provides methods ofinducing an immunological response in a host by administering to thehost an effective amount of an imidazoquinoline derived compound of thepresent disclosure, where the compound is capable of activating TLR 7 orcapable of dual activation of TLR7 and TLR8. Methods of the presentdisclosure also include methods of activating TLR7 by introducing acomposition including an imidazoquinoline derived compound of thepresent disclosure, such as a compound of Formula I and/or II to one ormore TLR7 receptors. In some embodiments the imidazoquinoline derivedcompound used to activate TLR7 is one or more of the imidazoquinolinederived compounds of Formula I and/or II described above or derivativesor analogues of each.

The present disclosure also provides methods of immunizing a host byadministering a vaccine including an antigen to the host, and alsoadministering to the host an adjuvant, where the adjuvant is animidazoquinoline derived compound of the present disclosure that iscapable of activating TLR7 or is capable of activating TLR7 and TLR8.The adjuvant may be administered in the same composition as the antigen,or they may be administered separately but at a similar time. Inembodiments of the methods of immunizing a host, the adjuvant compoundmay be chosen from, but is not limited to, compounds any of thecompounds listed above as having TLR7 and/or TLR7/TLR8 agonist activity.The adjuvant compound may be capable of activating TLR7 and TLR8, forinstance, compounds 7c, 7d, 19, 39, 35, 41, 52c, and derivatives andanalogues of each of these.

Thus, embodiments of the disclosure also include, a TLR7 agonistcomprising an imidazoquinoline derived compound chosen from compounds6c, 6d, 7c, 7d, 8, 12, 13, 14, 15, 16, 17, 18, 19, 21, 23, 25, 26-28,30b, 31, 33, 35, 37, 39, 60, 61, 62, 64, 65, derivatives thereof,analogues thereof, or pharmaceutically acceptable salts thereof.Embodiments of the present disclosure also include dual a TLR7/TLR8agonist comprising an imidazoquinoline derived compound chosen fromcompounds 7c, 7d, 19, 39, 35, 41, 52c, derivatives thereof, analoguesthereof, or pharmaceutically acceptable salts thereof. These agonistscan be used as vaccine adjuvants (alone, or as self-adjuvanting vaccineswhen coupled to an antigen), as probes (when coupled to or including areporter moiety), and the like, as described below.

Syntheses of Fluorescent Imidazoquinoline Conjugates as Probes of TLR7or TLR7/8

Further exploration on the imidazoquinoline chemotype led to discoveryof a highly active TLR7 and/or TLR7/8 dual agonistic molecule bearing afree primary amine on the N¹ substituent (e.g., compound 7d). Inembodiments, this compound was modified with a fluorescent reportermoiety to synthesize fluorescent imidazoquinoline analogues thatretained TLR7 and/or TLR7/8-agonistic activity, and were used to studythe distribution of TLR7 and also to examine its differential uptake inlymphocytic subsets. Details regarding the fluorescent imidazoquinolinederived compounds of the present disclosure, methods of making and usingsuch molecules are described in greater detail in Example 3 below.

The present disclosure thus describes probes for imaging activation ofTLR7, TLR8 or dual activation of TLR7 and 8. In embodiments, probes ofthe present disclosure include a TLR7 ligand or a dual TLR7/TLR8 ligandchosen from imidazoquinoline derived compounds of the present disclosurehaving a reporter moiety. The reporter moiety includes a reportermolecule capable of producing a detectable signal. In embodiments theTLR7 or dual TLR7/TLR8 ligand is chosen from compounds 7c, 7d, andderivatives and analogues of these compounds. In embodiments, compound7d was converted to an isothiocyanate derivative, compound 8, whichallows for coupling to amine-bearing fluorophores (such as but notlimited to bora-diazaindacene dye, and BODIPY-TR-cadaverine) to producea fluorescent imidazoquinoline analogue, such as compound 28. Inembodiments, the free primary amine on the N¹ substituent of 7d wascovalently coupled directly to commercially-available fluoresceinisothiocyanate and rhodamine B isothiocyanate, to produce fluorescentimidazoquinoline derived compounds 26 and 27. Compounds 26, 27, and 28were shown to retain TLR7 agonistic activity.

In embodiments, the reporter moiety for the probes of the presentdisclosure may include any reporter molecule capable of producing adetectable signal (e.g., optical, acoustic, magnetic, electrical, etc.),including, but not limited to, fluorescent molecules (e.g.,fluorophores, fluorescent dye, etc. as described above), MRI agents, andthe like. In embodiments the reporter molecule is a fluorescentcompound, such as in compounds 26, 27, and 28 as discussed in Example 3;in other embodiments the reporter molecule is biotin, such as incompound 37, discussed further in Example 6.

The present disclosure also provides methods of imaging activation ofTLR 7, TLR 8, or both TLR7 and TLR8 using the probes of the presentdisclosure. In embodiments of the methods of imaging activation of TLR7,TLR8 or TLR7/8, a sample including the receptors to be activated iscontacted with a probe of the present disclosure, and then thedetectable signal produced by the reporter molecule can be imaged byimaging methods and technology known to those of skill in the art.

Self-Adjuvanting Model Peptide, Protein and Polysaccharide Antigens withCovalently Bound TLR-7/8 Agonistic Imidazoquinolines

Embodiments of the present disclosure also include self-adjuvantingantigen/imidazoquinoline derived compounds. Such embodiments includeimidazoquinoline derived compounds of the present disclosure including apeptide or protein antigen moiety and/or a polysaccharide antigenmoiety. In embodiments, the imidazoquinoline derived compounds of thepresent disclosure, such as the TLR7 and TLR7/8 dual agonistic compoundsdescribed above, are used as a convenient precursor for the synthesis ofisothiocyanate derivatives (e.g., compound 8) and maleimide derivatives(e.g., compound 21), enabling direct conjugation to protein andpolysaccharide antigens to make self-adjuvanting vaccine constructs. Inan embodiment, the isothiocyanate derivative, 8, can be reacted withtri-glycine methyl ester model peptide to produce the adduct 29. In anembodiment, an isothiocyanate derivative (8) can be covalently coupledto a protein antigen, such as, but not limited to, α-lactalbumin, toproduce an embodiment of a self-adjuvanting α-lactalbumin construct.This compound induced robust, high-affinity immunoglobulin titers inmurine models of vaccination.

Additional embodiments of the present disclosure include maleimidederivatives of the imidazoquinoline derived compounds of the presentdisclosure, such as, but not limited to, compound 21. In embodiments,the maleimide derivative compound 21 can be coupled to glutathione toproduce the derivative compounds 30a and 30b. In an embodiment, themaleimide derivative, 21, can be covalently coupled to a peptide orprotein antigen moiety including an antigen, such as, but not limitedto, α-lactalbumin, to produce an embodiment of a self-adjuvantingα-lactalbumin construct. Additional details regarding theself-adjuvanting antigens with covalently bound TLR 7/8 dual agonisticmolecules, and methods of making and using such self-adjuvantingantigens are described in greater detail in Example 4 below. Whileα-lactalbumin represents one example of a protein or peptide antigenmoiety that can be coupled to the imidazoquinoline derived compounds ofthe present disclosure to produce self-adjuvanting peptide antigenimidazoquinoline derived compounds, other protein and peptide antigenscan be used within the scope of the present disclosure.

Polysaccharide antigens can also be covalently coupled toimidazoquinoline derived compounds of the present disclosure to providemidazoquinoline derived compounds having a polysaccharide antigenmoiety, as described in greater detail in Example 5 below. Inembodiments imidazoquinoline derived compounds of the present disclosureof Formula I or II, such as compound 7d can be coupled with apolysaccharide antigen, such as, but not limited to, maltoheptaose,which is used in a polysaccharide vaccine for N. meningitidis. In anembodiment compound 7d can be coupled with maltoheptaose to producecompound 31, which retained TLR7 activity. Embodiments ofself-adjuvanting polysaccharide antigens of the present disclosureinclude, but are not limited to, compound 31, described below.

The present disclosure also provides self-adjuvanting vaccines. Inembodiments, the self-adjuvanting vaccines include an antigen covalentlycoupled to an adjuvant compound of the present disclosure as describedabove. In embodiments the adjuvant compound can be a TLR7 agonist or adual TLR 7/8 agonist, such as the imidazoquinoline derived compounds asdescribed above. In embodiments, the adjuvant compounds areimidazoquinoline derived compounds that include a antigen moiety and/ora functionality for covalently coupling the antigen moiety, such as aprotein or peptide antigen (e.g., viral peptide) or a polysaccharideantigen (e.g., bacterial polysaccharide). For instance, the adjuvantcompound can be isothiocyanate derivatives and maleimide derivatives ofthe dual TLR 7/8 agonists of the present disclosure. In embodiments theadjuvant compound can be isothiocyanate derivatives and maleimidederivatives of a compound of Formula I or Formula II. In embodiments,the adjuvant compounds of the present disclosure include isothiocyanatederivatives and maleimide derivatives of compound 7d or derivatives oranalogues of 7d. In embodiments such compounds include but are notlimited to, compound 8 and compound 21. The isothiocyanate derivativesand maleimide derivatives of the adjuvant compounds of the presentdisclosure can be useful for covalently coupling peptide and proteinantigens.

The present disclosure also includes methods of immunizing a host byadministering a self-adjuvanting vaccine of the present disclosure tothe host. The self-adjuvanting vaccines may be useful in inducing anenhanced immune response from the host.

Tissue-Specific Imidazoquinoline Derived Compounds and TherapeuticMethods of Use

Embodiments of the present disclosure also include imidazoquinolinederived compounds modified with tissue-specific moieties to target theimidazoquinoline derived compounds to a specific location in a host. Inembodiments, the compounds of the present disclosure include a compoundof Formula I including a tissue-specific moiety that includes atissue-specific molecule/agent. In this manner, such tissue-specificimidazoquinoline derived compounds can also be used in methods oftreating certain conditions where targeted delivery to a specific regionin the host is useful. For instance, in an embodimentgalactosyl-terminating molecules can be used to target delivery ofcompounds to the liver of a host. Embodiments of the present disclosureinclude a derivatives and analogues of a compound of Formula I or II,such as 7d, modified with galactose to produce a liver-specificimidazoquinoline compound 33. Vitamins, such as, but not limited to,folic acid, biotin, and pyridoxal can be used to target compounds tocertain tumors. Thus, embodiments of the present disclosure includeimidazoquinoline derived compounds of the present disclosure modifiedwith vitamins, such as, but not limited to, folic acid, biotin andpyridoxal to produce tumor-specific compounds, such as folic acidderivative 35, biotin derivative 37, and pyridoxal derivative 39.

Embodiments of the present disclosure also include compositions fortreatment of a condition such as, but not limited to, hepatitis, chronicmyelogenous and hairy cell leukemias. In embodiments, such compositionsinclude an imidazoquinoline derived compound of the present disclosurein an amount effective to treat the condition. Interferon alpha is thecomposition of choice for treating conditions such as hepatitis;however, many patients do not tolerate oral administration of thecompound. Embodiments of the compounds of the present disclosure inducethe production of interferon alpha. When targeted to the host's liver,compounds of the present disclosure can induce production of interferonalpha by the patient's own liver, thus bypassing the need for oraladministration of interferon alpha. In an embodiment, imidazoquinolinederived compounds of the present disclosure, such as, but not limitedto, compounds of Formulas I and II, and derivatives and analogues ofsuch compounds can be modified by a tissue-specific agent to target thecompound to the host's liver. In an embodiment, the tissue-specificagent is galactose, which directs the compound to the liver of the host.Embodiments of such tissue-specific imidazoquinoline derived compoundsinclude, but are not limited to, compounds 33. Additional detailsregarding embodiments of tissue-specific imidazoquinoline derivedcompounds of the present disclosure are presented in Example 6, below.

In embodiments, imidazoquinoline derived compounds, such as, but notlimited to, compounds 33, 35, 37, and 39 and derivatives of each of theforegoing compounds or analogues thereof can be used to treat conditionssuch as hepatitis, chronic myelogenous and hairy cell leukemias. Inembodiments, compound 33, which directs the molecule to the liver of thehost, can be used to treat Hepatitis C. This targeted delivery helps toreduce side effects. The present disclosure also provides methods oftreatment of conditions such as, but not limited to, hepatitis, chronicmyelogenous and hairy cell leukemias, including administering to a hostin need of treatment for the condition and effective amount of animidazoquinoline derived compound described immediately above. Thus, inembodiments the present disclosure includes a method of treatment of acondition chosen from hepatitis, chronic myelogenous and hairy cellleukemia comprising administering to a host in need of treatment for thecondition an effective amount of an imidazoquinoline derived compound ofFormula I or II, above, coupled to a tissue-specific agent selected fromgalactose, folic acid, biotin, or pyridoxal.

Additional methods of the present disclosure include methods of inducingproduction of interferon alpha in a host by administering to the host aneffective amount of an imidazoquinoline derived compound of the presentdisclosure. In embodiments, the imidazoquinoline derived compound can bechosen from, but is not limited to, compound 33 and derivatives oranalogues thereof.

The embodiments of tissue-specific imidazoquinoline derived compoundsinclude, described above are merely representative examples, and theimidazoquinoline derived compounds of the present disclosure may bemodified with many other tissue-specific compounds to achieve targeteddelivery to various tissues and/or locations in a host.

Dendrimers and Dimers of Imidazoquinoline Derived Compounds

The present disclosure also includes dendrimers of the imidazoquinolinederived compounds of the present disclosure, and methods of making andusing the dendrimers, including, but not limited to, trimers andhexamers of the imidazoquinoline derived compounds of the presentdisclosure. In embodiments, the present disclosure includes dendrimers(e.g., trimers and/or hexamers) of the imidazoquinoline derivedcompounds of Formula I or Formula II.

The larger size of dendrimers bearing three or six units of animidazoquinoline derived compound of the present disclosure may extendactivity of the compounds by slowing diffusion of the compounds.Embodiments of the present disclosure include dendrimers ofimidazoquinoline derived compounds of Formula I and/or Formula II.Embodiments include dendrimers of compound 7d, such as, but not limitedto, the trimeric imidazoquinoline dendrimer 41 and the hexamericimidazoquinoline dendrimer 43.

In embodiments, trimers of the imidazoquinoline derived compounds of thepresent disclosure can be made and retain TLR7 and/or TLR7/8 agonisticactivity. Embodiments including trimers and hexamers of compound 7d aredescribed in greater detail in Example 7 below. In embodiments, trimersof imidazoquinoline derived compounds of the present disclosure includecompound 41. In embodiments, hexamers of imidazoquinoline derivedcompounds of the present disclosure include compound 43, which retainsTLR7 activity.

The present disclosure also includes dimeric imidazoquinoline variantsof the imidazoquinoline derived compounds of the present disclosure.Embodiments of the disclosure include, but are not limited to, dimers ofthe imidazoquinoline derived compounds of Formulas I and/or II with theunits linked at different positions, such as, but not limited to, theN¹-(4-aminomethylene)benzyl-linked dimers 52a-c, and the C8-NH₂ linkeddimers 55a-c. In embodiments, the present disclosure includes dimers ofvariants of compound 7d, with the units linked at different positions,such as compounds 52a-c. Dimeric compounds 51a-d, 52a-c, and 55-a-cdemonstrated TLR7 agonistic activity, and 55c demonstrated dualTLR7/TLR8 activity. Thus, the present disclosure includes TLR7 and/ordual TLR7/TLR8 agonists including dimeric compound derivatives ofFormulas I and II, such as, but not limited to, the compounds 52a-c, and55-a-c.

Embodiments of dimeric imidazoquinoline derived compounds of the presentdisclosure also include, but are not limited to, C2-linked compounds47a-b, 49a-b, the C4-NH₂ linked dimers 51a-d. Dimeric compounds 47a-b,49a-b can be represented by the generic structure of Formula III, below.

where, R₁ is independently selected from the group consisting ofhydrogen, halogen (e.g., a group including —Cl, —Br, —F), nitro (e.g., agroup including —NO₂), —NH₂, azido (e.g., a group including —N₃),hydroxyl (e.g., a group including —OH), —CF₃; R₃ is independentlyselected from the group consisting of hydrogen or —(CH₂)_(x)—NH₂, and xis any integer form 1 to 10. The dimeric imidazoquinoline derivedcompounds of Formula III, such as, but not limited to, 47a-b, and 49a-bdemonstrated unexpected TLR7 antagonistic behavior, in contrast to theagonistic activity of the dimers based on the compounds of Formulas Iand II, such as dimeric compounds 52a-c and 55a-c. Thus, in embodiments,the present disclosure includes TLR7 antagonists including compounds ofFormula III, derivatives thereof, analogues thereof, andpharmaceutically acceptable salts thereof.

Additional details regarding embodiments of the dimers of theimidazoquinoline derived compounds of the present disclosure aredescribed below in Example 8.

Dual TLR2/TLR7 Adjuvants

Embodiments of the present disclosure also include imidazoquinolinederived compounds with TLR7 agonistic activity including a TLR2 agonistmoiety to provide dual TLR2/TLR7 adjuvant compounds. In embodiments,TLR7 activating imidazoquinoline derived compounds of the presentdisclosure described above, such as those of Formula I or II (e.g.,compounds 7d, 8, 12, 13, 14, 15, etc.) or their derivatives andanalogues are coupled to a TLR2 agonist moiety that includes a TLR2agonist compound, such as, but not limited to,S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cysteinyl-S-serine (PAM(2)CS)compounds and derivatives of PAM(2) CS compounds. Embodiments of dualTLR2/TLR7 adjuvant compounds include, but are not limited to,thiourea-linked hybrid compounds 60-65, and derivatives and analogues ofthese compounds. Additional details regarding the dual TLR2/TLR7adjuvant compounds of the present disclosure are described in Example 9below.

Additional details regarding the imidazoquinoline derived compounds ofthe present disclosure, probes and adjuvants comprising theimidazoquinoline derived compounds, compositions comprising theimidazoquinoline derived compounds, methods of making theimidazoquinoline derived compounds of the present disclosure, andmethods of using the imidazoquinoline derived compounds of the presentdisclosure can be found in the following Examples.

The specific examples below are to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentdisclosure to its fullest extent. All publications recited herein arehereby incorporated by reference in their entirety.

It should be emphasized that the embodiments of the present disclosure,particularly, any “preferred” embodiments, are merely possible examplesof the implementations, merely set forth for a clear understanding ofthe principles of the disclosure. Many variations and modifications maybe made to the above-described embodiment(s) of the disclosure withoutdeparting substantially from the spirit and principles of thedisclosure. All such modifications and variations are intended to beincluded herein within the scope of this disclosure, and protected bythe following embodiments.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions and compounds disclosedherein. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is at or nearatmospheric. Standard temperature and pressure are defined as 20° C. and1 atmosphere.

It should be noted that ratios, concentrations, amounts, and othernumerical data may be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a concentration range of “about0.1% to about 5%” should be interpreted to include not only theexplicitly recited concentration of about 0.1 wt % to about 5 wt %, butalso include individual concentrations (e.g., 1%, 2%, 3%, and 4%) andthe sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within theindicated range. In an embodiment, the term “about” can includetraditional rounding according to significant figures of the numericalvalue.

EXAMPLES

Now having described the embodiments of the present disclosure, ingeneral, the following Examples describe some additional embodiments ofthe present disclosure. While embodiments of present disclosure aredescribed in connection with the following examples and thecorresponding text and figures, there is no intent to limit embodimentsof the present disclosure to this description. On the contrary, theintent is to cover all alternatives, modifications, and equivalentsincluded within the spirit and scope of embodiments of the presentdisclosure

Toll-like receptors (TLRs), of which 10 are known in the human genome,are pattern recognition receptors.^(1,2) The ligands for these receptorsare highly conserved microbial molecules. The activation of TLRs bytheir cognate ligands leads to activation of innate immune effectormechanisms, including the production of pro-inflammatory cytokines, andup-regulation of MHC molecules and co-stimulatory signals inantigen-presenting cells as well as activating natural killer (NK)cells. The consequence of activation of the innate immune systemmobilizes and amplifies specific adaptive immune responses involvingboth T- and B-cell effector functions.⁵⁻⁷ Thus, TLR stimuli serve tolink innate and adaptive immunity⁵ and can therefore be exploited aspowerful adjuvants in eliciting both primary and anamnestic immuneresponses.

Representative members of virtually the entire compendium of known TLRagonists have been examined in a series of hierarchical assays includingprimary TLR-reporter assays, secondary indices of immune activation suchas cytokine induction and activation of lymphocytic subsets in wholehuman blood, and tertiary screens characterizing transcriptomalactivation patterns with a view to identifying optimal immunostimulatorychemotypes.⁸ Of all the innate immune stimuli examined, TLR7 agonistswere found to be extraordinarily immunostimulatory, stimulatingvirtually all subsets of lymphocytes, and yet without inducing dominantproinflammatory cytokine responses (unlike TLR4-5 or -8 agonists, whichwere proinflammatory and therefore may exert systemic toxicity).⁸TLR7-active compounds therefore represent candidates as potentialvaccine adjuvants and immune response modifiers. In some embodiments isalso desirable to identify compounds capable of dual activation of TLR7and TLR8, as discussed in grater detail below.

Long before endosomal TLR7 was discovered to serve as the primary sensorfor short, single-stranded, GU-rich RNA sequences (ssRNA), mainly ofviral origin,⁹⁻¹¹ a number of small molecules were synthesized andevaluated in the 1970s and '80s for antiviral activities owing to theirpronounced Type I interferon (IFN-α and -β) inducing properties.¹²⁻¹⁶The 1H-imidazo[4,5-c]quinolines were found to be good Type I IFNinducers in human cell-derived assays,¹⁷ and FDA approval was obtainedin 1997 for Imiquimod (1, FIG. 1) for the treatment of basal cellcarcinoma and actinic keratosis, and Gardiquimod, 2 (FIG. 1), is a knownimidazoquinoline TLR7 agonist.¹⁸

It was not until 2002, however, that the mechanistic basis of IFNinduction by the imidazoquinolines was found to be a consequence of TLR7engagement and activation.¹⁹ Other than the original studies, performedby investigators at 3M Pharmaceuticals,¹⁷ structure-activityrelationships of the imidazoquinoline chemotype remains poorly explored,perhaps attributable in part to recent interest in the 8-hydroxy-adeninecompounds as alternate TLR7-agonists,²⁰⁻²³ which appear to lack emeticside-effects observed in ferrets upon oral administration.²² A positivefactor related to imidazoquinolines is that imiquimod is alreadyapproved for topical use. The present example describes the discovery ofa novel and unique compound, 7d (Scheme 1), and its derivatives andanalogues, which have not been described hitherto in the literature.This compound has served as a core structure for the syntheses ofseveral imidazoquinoline derived compounds described herein.

Materials and Methods:

All of the solvents and reagents used were obtained commercially andused as such unless noted otherwise. Moisture- or air-sensitivereactions were conducted under nitrogen atmosphere in oven-dried (120°C.) glass apparatus. The solvents were removed under reduced pressureusing standard rotary evaporators. Flash column chromatography wascarried out using RediSep Rf ‘Gold’ high performance silica columns onCombiFlash Rf instruments unless otherwise mentioned, while thin-layerchromatography was carried out on silica gel CCM pre-coated aluminumsheets. Purity for all final compounds was confirmed to be greater than97% by LC-MS using a Zorbax Eclipse Plus 4.6 mm×150 mm, 5 μm analyticalreverse phase C₁₈ column with H₂O-isopropanol or H₂O—CH₃CN gradients andan Agilent ESI-TOF mass spectrometer (mass accuracy of 3 ppm) operatingin the positive ion acquisition mode. All the compounds synthesized wereobtained as solids.

NF-κB induction: The induction of NF-κB was quantified using HEK-Blue-7cells and HEK-Blue-8 cells as previously described by us.^(8,24) HEK293cells were stably transfected with human TLR7 (or human TLR8), MD2, andsecreted alkaline phosphatase (sAP), and were maintained in HEK-Blue™Selection medium containing zeocin and normocin. Stable expression ofsecreted alkaline phosphatase (sAP) under control of NF-κB/AP-1promoters is inducible by the TLR7 (or TLR8) agonists, and extracellularsAP in the supernatant is proportional to NF-κB induction. HEK-Bluecells were incubated at a density of ˜10 cells/ml in a volume of 80μL/well, in 384-well, flat-bottomed, cell culture-treated microtiterplates until confluency was achieved, and subsequently gradedconcentrations of stimuli. sAP was assayed spectrophotometrically usingan alkaline phosphatase-specific chromogen (present in HEK-detectionmedium as supplied by the vendor) at 620 nm.

Example 1 Synthesis and Activity of N¹-Substituted Imidazoquinolines

To a solution of 3 (100 mg, 0.41 mmol) in 5 mL of anhydrousdichloromethane, were added triethylamine (54 mg, 0.53 mmol) andnaphthalen-1-ylmethanamine (71 mg, 0.45 mmol). The reaction mixture wasrefluxed at 45° C. for 30 minutes. The solvent was then evaporated undervacuum and product was isolated using column chromatography to obtainthe intermediate compound 4a. To a solution of 4a in 10 mL of EtOAc,were added a catalytic amount of Pt/C and Na₂SO₄. The reaction mixturewas subjected to hydrogenation at 55 psi hydrogen pressure for 4 hours.The reaction mixture was then filtered through celite and the filtratewas evaporated under vacuum to obtain compound 5a (90 mg). To a solutionof 5a (90 mg, 0.27 mmol) in anhydrous THF, were added triethylamine (41mg, 0.41 mmol) and valeryl chloride (39 mg, 0.32 mmol). The reactionmixture was stirred at room temperature for 6 hours. The solvent wasthen removed under vacuum, and the residue was dissolved in ethylacetate and washed with water. The ethyl acetate fraction was then driedusing Na₂SO₄ and evaporated under vacuum to obtain the intermediateamide compound, which was then dissolved in 2 mL of 2M solution ofammonia in MeOH. The sealed reaction vessel was heated 150° C. for 24hours. The solvent was then removed under vacuum and the residue waspurified using column chromatography (7% MeOH/dichloromethane) to obtaincompound 6a (62 mg; 40%). ¹H NMR (500 MHz, DMSO) δ 8.40 (d, J=8.3 Hz,1H), 8.05 (d, J=8.1 Hz, 1H), 7.86 (d, J=8.2 Hz, 1H), 7.77 (t, J=7.2 Hz,1H), 7.69 (t, J=7.5 Hz, 1H), 7.62 (d, J=8.2 Hz, 1H), 7.47 (d, J=8.2 Hz,1H), 7.33 (t, J=7.5 Hz, 1H), 7.27 (t, J=7.7 Hz, 1H), 7.08 (s, 2H), 6.90(t, J=7.5 Hz, 1H), 6.39 (d, J=7.1 Hz, 1H), 6.35 (s, 2H), 2.92 (t, J=7.6Hz, 2H), 1.69 (dt, J=15.2, 7.6 Hz, 2H), 1.32 (dt, J=14.6, 7.4 Hz, 2H),0.80 (t, J=7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO) b 154.39, 151.16,133.62, 133.25, 131.77, 129.64, 128.74, 127.88, 126.91, 126.80, 126.48,126.15, 125.58, 123.07, 121.57, 121.42, 119.87, 114.11, 46.60, 29.45,26.08, 21.70, 13.61. MS (ESI) calculated for C₂₅H₂₄N₄, m/z 380.20, found381.21 (M+H)⁺.

Compound 6b was synthesized similarly as described for compound 6a.

6b: 1-(Biphenyl-4-ylmethyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine

¹H NMR (500 MHz, CDCl₃) δ 7.88 (d, J=7.9 Hz, 1H), 7.78-7.73 (m, 1H),7.60-7.56 (m, 2H), 7.55-7.52 (m, 2H), 7.51-7.47 (m, 1H), 7.45-7.40 (m,2H), 7.35 (ddt, J=8.5, 6.5, 1.4 Hz, 1H), 7.30-7.25 (m, 1H), 7.10 (d,J=8.3 Hz, 2H), 5.81 (s, 2H), 2.94-2.91 (m, 2H), 1.84 (dt, J=15.4, 7.6Hz, 2H), 1.51-1.41 (m, 2H), 0.95 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz,CDCl₃) δ 156.27, 149.52, 141.25, 139.59, 135.17, 132.65, 129.02, 128.64,127.93, 127.52, 126.72, 125.52, 124.77, 124.58, 120.31, 120.23, 112.54,48.72, 29.17, 26.80, 22.15, 13.47. MS (ESI) calculated for C₂₇H₂₆N₄, m/z406.22, found 407.22 (M+H)⁺.

Synthesis of Compound 7c:1-(3-(Aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine

To a solution of 3 (200 mg, 0.83 mmol) in 5 mL of anhydrousdichloromethane, were added triethylamine (92 mg, 0.91 mmol) andtert-butyl 3-(aminomethyl)benzylcarbamate (215 mg, 1.06 mmol) dissolvedin 2 mL of anhydrous MeOH. The reaction mixture was refluxed at 45° C.for 30 minutes. The solvent was then evaporated under vacuum and productwas isolated using column chromatography to obtain the intermediatecompound 4c. To a solution of 4c in 10 mL of EtOAc, were added acatalytic amount of Pt/C and Na₂SO₄. The reaction mixture was subjectedto hydrogenation at 55 psi hydrogen pressure for 4 hours. The reactionmixture was then filtered through celite and the filtrate was evaporatedunder vacuum to obtain compound 5c (202 mg). To a solution of 5c (202mg, 0.49 mmol) in anhydrous THF, were added triethylamine (64 mg, 0.64mmol) and valeryl chloride (73 mg, 0.54 mmol). The reaction mixture wasstirred at room temperature for 6 hours. The solvent was then removedunder vacuum, and the residue was dissolved in ethyl acetate and washedwith water. The ethyl acetate fraction was then dried using Na₂SO₄ andevaporated under vacuum to obtain the intermediate amide compound, whichwas then dissolved in 2 mL of 2M solution of ammonia in MeOH. The sealedreaction vessel was heated 150° C. for 24 hours. The solvent was thenremoved under vacuum and the residue was purified using columnchromatography (9% MeOH/dichloromethane) to obtain compound 6c (44 mg;12%). This was then dissolved in 10 mL of HCl/dioxane solution andstirred for 12 hours. The solvent was then removed to obtain compound 7c(52 mg, 15%). ¹H NMR (500 MHz, MeOD) δ 7.85 (s, 1H), 7.67 (d, J=7.0 Hz,1H), 7.52 (s, 1H), 7.39-7.18 (m, 4H), 7.02 (s, 1H), 5.92 (s, 2H), 4.01(s, 2H), 2.94 (s, 2H), 1.80 (s, 2H), 1.41 (d, J=4.4 Hz, 2H), 0.88 (t,J=6.1 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 159.02, 150.28, 137.51, 135.84,135.26, 131.34, 131.06, 129.95, 127.66, 127.46, 126.67, 125.73, 123.01,119.66, 114.08, 50.24, 44.18, 30.32, 27.98, 23.45, 14.25. MS (ESI)calculated for C₂₂H₂₅N₅, m/z 359.21, found 360.22 (M+H)⁺.

Compound 7d was synthesized similarly as described for compound 7c.

7d: 1-(4-(Aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine

¹H NMR (500 MHz, MeOD) δ 7.85 (d, J=8.2 Hz, 1H), 7.69 (d, J=8.3 Hz, 1H),7.54 (t, J=7.7 Hz, 1H), 7.40 (d, J=7.7 Hz, 2H), 7.26 (t, J=7.6 Hz, 1H),7.10 (d, J=7.8 Hz, 2H), 5.93 (s, 2H), 4.01 (s, 2H), 2.94 (t, J=7.6 Hz,2H), 1.83-1.71 (m, 2H), 1.43-1.32 (m, 2H), 0.86 (t, J=7.3 Hz, 3H). ¹³CNMR (126 MHz, MeOD) δ 159.02, 150.27, 137.51, 137.47, 135.33, 134.59,131.17, 131.11, 127.54, 126.51, 125.53, 122.95, 119.66, 114.03, 49.93,43.81, 30.31, 27.78, 23.35, 14.12. MS (ESI) calculated for C₂₂H₂₅N₅, m/z359.21, found 360.22 (M+H)⁺.

TLR7 and TLR8 agonistic activity of compound 6a, 6b, 7c, and 7d weretested, and results are shown in FIGS. 2A (TLR7 activity) and 2B (TLR8activity). The N¹-naphthylenemethyl-substituted compound 6a wasinactive, and the N¹-biphenyl-4-methyl compound 6b was weakly active(EC₅₀: 396 nM); the N¹-(4-aminomethyl)benzyl substituted analogue 7d wasthe most active compound and served as a core structure for thesyntheses of many other imidazoquinoline derived compounds describedbelow. (EC₅₀: 20 nM, FIGS. 2A and 2B). 7d was more active than itsN¹-(3-aminomethyl)benzyl regioisomer 7c (EC₅₀: 110 nM, FIGS. 2A-2B).

Imidazoquinoline Derived Compounds

From compound 7d, a variety of imidazoquinoline derived compounds of thepresent disclosure, represented by FORMULA I, below, were synthesized asdescribed in greater detail below.

In embodiments of the imidazoquinoline derived compounds of the presentdisclosure of Formula I,

R is selected from the group consisting of: —NH(R₅) or isothiocyanate(—NCS);

R₅ is selected from the group consisting of hydrogen (—H), acetyl (e.g.,a group that includes —COCH₃), —CO-tert-Bu (-Boc), —CO—(CH₂)_(x)—R₆,C₁-C₁₆ alkyl, —CO-4-(phenylboronic acid),—C(S)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH₂,

a reporter moiety, a tissue-specific moiety, a peptide antigen moiety, aprotein antigen moiety, a polysaccharide antigen moiety, and a TLR2agonist moiety;

R₆ is selected from the group consisting of hydrogen (—H), alkyne (e.g.,a group that includes a carbon-carbon triple bond such as —C≡CH), azido(—N₃), carboxylic acid (e.g., a group that includes a —CO₂H),—CONH—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—R₇;

R₇ is selected from the group consisting of amino (e.g., a group thatincludes a —NH₂), isothiocyanate (e.g., a group that includes a —NCS) or—NH—CO—(CH₂)_(x)—CO₂H;

R₈ is selected from a peptide antigen moiety or a protein antigenmoiety; and

x is any integer from 1 to 10.

Embodiments of compounds of Formula I and derivatives of such compoundswere synthesized and tested including, but not limited to, compounds 6d,7d, 8, 12, 13, 14, 15, 16, 17, 18, 19, 21, 23, 25, 26, 27, 28, 29, 30a,30b, 31, 33, 35, 37, 39, 60, 61, 62, 63, 64, and 65, which are describedin detail in the Examples below.

Furthermore, as described in Scheme 1, and as shown by the exemplarycompounds 7c and 7d, a variety of compounds can be synthesized as shownby the representative structure of Formula II, below, whereinsubstituents shown on the N1-benzyl unit could be independently at theortho, meta, or para positions, and the R groups are as defined below.

where,

R₁ and R₃ are each independently selected from the group consisting ofhydrogen, halogen (e.g., a group that includes —Cl, —Br, —F), nitro(e.g., a group that includes —NO₂), —NH₂, azido (e.g., a group thatincludes —N₃), hydroxyl (e.g., a group that includes —OH), —CF₃,carboxylic acid (e.g., a group that includes —CO₂H) or —CO₂R₂;

R₂ is a C₂-C₅ alkyl, and

R₄ selected from the group consisting of: —NH(R₅) or isothiocyanate(—NCS);

R₅ is selected from the group consisting of hydrogen (—H), acetyl (e.g.,a group that includes —COCH₃), —CO-tert-Bu (-Boc), —CO—(CH₂)_(x)—R₆,C₁-C₁₆ alkyl, —CO-4-(phenylboronic acid),—C(S)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH₂,

a reporter moiety, a tissue-specific moiety, a peptide antigen moiety, aprotein antigen moiety, a polysaccharide antigen moiety, and a TLR2agonist moiety;

R₆ is selected from the group consisting of hydrogen (—H), alkyne (e.g.,a group that includes a carbon-carbon triple bond such as —C≡CH), azido(—N₃), carboxylic acid (e.g., a group that includes a —CO₂H),—CONH—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—R₇;

R₇ is selected from the group consisting of amino (e.g., a group thatincludes a —NH₂), isothiocyanate (e.g., a group that includes a —NCS) or—NH—CO—(CH₂)_(x)—CO₂H;

R₈ is selected from a peptide antigen moiety or a protein antigenmoiety; and

x is any integer from 1 to 10.

Embodiments of compounds of Formula II, and derivatives of suchcompounds were synthesized and tested as described in detail in theseExamples.

Example 2 Derivatives of1-(4-(Aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (7d)

Synthesis of Compound 8:2-Butyl-1-(4-(isothiocyanatomethyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine

To a solution of 7d (150 mg, 0.35 mmol) in anhydrous dichloromethane,were added carbon disulfide (266 mg, 3.5 mmol) and triethylamine (106mg, 1.05 mmol). The reaction mixture was stirred for an hour and thenwas cooled to 0° C. Di-tert-butyl dicarbonate (76 mg, 0.35 mmol) and acatalytic amount of DMAP were added to the reaction mixture. Thereaction mixture was stirred for 18 hours and then the solvent wasremoved under vacuum. The residue was purified using columnchromatography (7% MeOH/dichloromethane) to obtain compound 8 (105 mg,75%). ¹H NMR (400 MHz, CDCl₃) δ 7.87-7.83 (m, 1H), 7.68 (dd, J=8.3, 0.8Hz, 1H), 7.51-7.45 (m, 1H), 7.32 (d, J=8.2 Hz, 2H), 7.23-7.17 (m, 1H),7.10 (d, J=8.2 Hz, 2H), 6.52 (s, 2H), 5.78 (s, 2H), 4.71 (s, 2H),2.94-2.86 (m, 2H), 1.82 (dt, J=15.5, 7.6 Hz, 2H), 1.52-1.41 (m, 2H),0.95 (t, J=7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 155.19, 150.27,135.10, 134.65, 134.57, 128.20, 127.95, 126.10, 125.92, 124.08, 123.59,119.94, 113.99, 48.74, 48.21, 29.71, 27.12, 22.47, 13.76. MS (ESI)calculated for C₂₃H₂₃N₅S, m/z 401.17, found 402.18 (M+H)⁺.

Synthesis of Compound12:1-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)phenyl)-3,7,23-trioxo-12,15,18-trioxa-2,8,22-triazaheptacosan-27-oicacid

To a solution of compound 9 (200 mg, 0.91 mmol) in anhydrous THF, wereadded triethylamine (320 μL, 2.28 mmol) and glutaric anhydride (212 mg,1.86 mmol) and the reaction mixture was stirred for 30 min. The solventwas then removed under vacuum to obtain the compound 10 in quantitativeyields. To a solution of compound 10 (100 mg, 0.15 mmol) in anhydrousDMF, were added triethylamine (53 μL, 0.38 mmol), HBTU (64 mg, 0.17mmol) and 7d (63 mg, 0.15 mmol). The reaction mixture was then stirredfor 6 hours, followed by removal of the solvent under vacuum. Theresidue was then purified using column chromatography to obtain thecompound 12 (37 mg, 32%). MS (ESI) calculated for C₄₂H₅₉N₇O₈, m/z789.4425, found 790.4513 (M+H)⁺.

Synthesis of Compound 13:5-((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)amino)-5-oxopentanoicacid

To a solution of compound 7d (50 mg, 0.12 mmol) in anhydrous THF, wereadded triethylamine (30 μL, 0.29 mmol) and glutaric anhydride (13 mg,0.12 mmol) and the reaction mixture was stirred for 30 min. The solventwas then removed under vacuum to obtain the triethylammonium salt ofcompound 13 in quantitative yields. ¹H NMR (400 MHz, DMSO) δ 7.91 (d,J=8.4 Hz, 1H), 7.73 (d, J=8.2 Hz, 1H), 7.54 (t, J=7.7 Hz, 1H), 7.27 (t,J=7.6 Hz, 1H), 7.20 (d, J=8.0 Hz, 2H), 7.01 (d, J=8.0 Hz, 2H), 5.91 (s,2H), 5.75 (s, 1H), 4.20 (s, 2H), 4.12 (s, 1H), 3.07 (q, J=7.3 Hz, 12H),2.94 (t, J=7.7 Hz, 2H), 2.30-2.09 (m, 4H), 1.78-1.64 (m, 4H), 1.38 (dd,J=14.9, 7.4 Hz, 2H), 1.20 (t, J=7.3 Hz, 17H), 0.87 (t, J=7.3 Hz, 3H). MS(ESI) calculated for C₂₇H₃₁N₅O₃, m/z 473.2427, found 474.2551 (M+H)⁺.

Synthesis of Compound 14:N¹-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-N⁵-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)glutaramide

To the solution of compound 9 (500 mg, 2.3 mmol) in anhydrousdichloromethane was added, di-tert-butyl dicarbonate (454 mg, 2.08 mmol)and the reaction was stirred for 1 hour, followed by removal of thesolvent under vacuum. To the residue dissolved in anhydrous THF wereadded, triethylamine (52 mg, 5.2 mmol) and glutaric anhydride (445 mg,3.9 mmol). The reaction mixture was stirred for 2 hours followed byremoval of the solvent under vacuum to obtain the crude residue whichwas purified using column chromatography (20% MeOH/dichloromethane) toyield compound 11 (400 mg, 41%). To a solution of 11 (125 mg, 0.23 mmol)in anhydrous DMF were added, triethylamine (60 mg, 0.59 mmol), HBTU (98mg, 0.26 mmol), 7d (100 mg, 0.23 mmol) and a catalytic amount of DMAPsequentially. The reaction mixture was stirred for 12 hours followed byremoval of the solvent under vacuum to obtain the residue which waspurified using column chromatography (20% MeOH/CH₂Cl₂) to obtain theN-Boc protected intermediate which was N-Boc deprotected by stirring in1 mL of 4M HCl/dioxane solution for 6 hours followed by removal of thesolvent under vacuum to obtain hydrochloride salt of compound 14 (140mg, 80%). 1H NMR (500 MHz, MeOD) δ 7.96 (dd, J=8.4, 0.7 Hz, 1H), 7.75(dd, J=8.4, 0.8 Hz, 1H), 7.63 (ddd, J=8.4, 7.3, 1.2 Hz, 1H), 7.36 (ddd,J=8.4, 7.3, 1.1 Hz, 1H), 7.27 (d, J=8.3 Hz, 2H), 7.05 (d, J=8.2 Hz, 2H),5.93 (s, 2H), 4.32 (s, 2H), 3.67-3.59 (m, 8H), 3.59-3.54 (m, 2H), 3.49(t, J=6.1 Hz, 2H), 3.22 (t, J=7.0 Hz, 2H), 3.08 (t, J=6.4 Hz, 2H),3.02-2.96 (m, 2H), 2.21 (dt, J=21.1, 7.6 Hz, 4H), 1.94-1.80 (m, 6H),1.73 (dd, J=13.4, 6.5 Hz, 2H), 1.45 (dd, J=15.0, 7.5 Hz, 2H), 0.94 (t,J=7.4 Hz, 3H). 13C NMR (126 MHz, MeOD) δ 175.35, 175.26, 159.05, 150.46,140.34, 137.61, 135.34, 135.23, 130.95, 129.50, 126.84, 126.42, 125.88,122.97, 119.60, 114.22, 71.39, 71.09, 71.05, 71.00, 70.39, 69.65, 49.83,43.54, 40.17, 37.66, 36.31, 36.20, 30.50, 30.34, 28.05, 27.76, 23.32,23.28, 14.10. MS (ESI) calculated for C₃₇H₅₃N₇O₅, m/z 675.41, found676.4270 (M+H)⁺ and 338.7178 (M+2H)⁺².

Synthesis of Compound 15:N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-N⁵-(3-(2-(2-(3-isothiocyanatopropoxy)ethoxy)ethoxy)propyl)glutaramide

To a solution of 14 (140 mg, 0.19 mmol) in anhydrous dichloromethane,were added carbon disulfide (143 mg, 1.89 mmol) and triethylamine (47mg, 0.469 mmol). The reaction mixture was stirred for an hour.Di-tert-butyl dicarbonate (41 mg, 0.19 mmol) and a catalytic amount ofDMAP were added to the reaction mixture. The reaction mixture wasstirred for 18 hours and then the solvent was removed under vacuum. Theresidue was purified using column chromatography (20% MeOH/CH₂Cl₂) toobtain the compound 15 (55 mg, 40%). MS (ESI) calculated forC₃₈H₅₁N₇O₅S, m/z 717.3672, found 718.3578. (M+H)⁺.

Synthesis of Compound 16:N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide

To a solution of compound 7d (50 mg, 0.116 mmol) in anhydrous THF, wereadded triethylamine (35 mg, 0.35 mmol) and palmitoyl chloride (35 mg,0.13 mmol). The reaction mixture was stirred for 1 hour, followed byremoval of the solvent under vacuum. The residue was then dissolved inethylacetate and washed with water, brine, dried using sodium sulfateand concentrated under vacuum to obtain the residue which was purifiedusing column chromatography (8% MeOH/dichloromethane) to obtain compound16 (25 mg, 36%). ¹H NMR (500 MHz, CDCl₃) δ 7.81 (dd, J=8.4, 0.7 Hz, 1H),7.68 (dd, J=8.3, 0.9 Hz, 1H), 7.44 (ddd, J=8.4, 7.1, 1.3 Hz, 1H), 7.24(d, J=8.2 Hz, 2H), 7.16 (ddd, J=8.3, 7.2, 1.2 Hz, 1H), 6.99 (d, J=8.2Hz, 2H), 6.40 (s, 2H), 5.81 (t, J=5.7 Hz, 1H), 5.71 (s, 2H), 4.40 (d,J=5.9 Hz, 2H), 2.91-2.81 (m, 2H), 2.23-2.11 (m, 2H), 1.85-1.74 (m, 2H),1.67-1.57 (m, 2H), 1.49-1.38 (m, 2H), 1.34-1.16 (m, 24H), 0.93 (t, J=7.4Hz, 3H), 0.87 (t, J=7.0 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 173.33,155.29, 150.54, 139.05, 134.81, 134.10, 128.88, 128.22, 126.14, 125.95,124.50, 123.58, 120.24, 114.35, 48.96, 43.12, 36.95, 32.12, 29.92,29.90, 29.89, 29.88, 29.86, 29.84, 29.81, 29.68, 29.56, 29.53, 29.52,27.30, 25.92, 22.89, 22.67, 14.33, 13.96. MS (ESI) calculated forC₃₈H₅₅N₅O, m/z 597.44, found 598.45 (M+H)⁺.

Synthesis of Compound 17:2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine

To a solution of compound 7d (50 mg, 0.116 mmol) in anhydrous DMF, wereadded triethylamine (35 mg, 0.35 mmol) and iodohexadecane (41 mg, 0.116mmol). The reaction mixture was stirred for 18 hours followed by removalof the solvent under vacuum. The residue was then dissolved inethylacetate and washed with saturated sodium bicarbonate solution,water, brine, dried using sodium sulfate and concentrated under vacuumto obtain the residue which was purified using column chromatography(15% MeOH/dichloromethane) to obtain compound 17 (11 mg, 16%). ¹H NMR(500 MHz, MeOD) δ 7.94 (dd, J=8.4, 0.7 Hz, 1H), 7.76 (dd, J=8.4, 0.8 Hz,1H), 7.63 (ddd, J=8.4, 7.3, 1.2 Hz, 1H), 7.52 (d, J=8.3 Hz, 2H), 7.34(ddd, J=8.4, 7.3, 1.1 Hz, 1H), 7.20 (d, J=8.3 Hz, 2H), 6.01 (s, 2H),4.17 (s, 2H), 3.03-2.95 (m, 4H), 1.87-1.81 (m, 2H), 1.70-1.63 (m, 2H),1.47 (dd, J=15.0, 7.5 Hz, 2H), 1.37-1.24 (m, 26H), 0.94 (t, J=7.4 Hz,3H), 0.89 (t, J=7.0 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 159.19, 150.63,138.26, 137.68, 135.47, 132.90, 132.19, 131.14, 127.71, 126.54, 126.14,123.02, 119.77, 114.31, 51.86, 33.23, 30.95, 30.94, 30.92, 30.88, 30.77,30.63, 30.62, 30.51, 30.31, 27.91, 27.73, 27.25, 23.89, 23.47, 14.59,14.26. MS (ESI) calculated for C₃₈H₅₇N₅, m/z 583.46, found 584.47(M+H)⁺.

Synthesis of Compound 18:N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-azidoacetamide

To a solution of 1-bromo acetic acid (52 mg, 0.37 mmol) in anhydrous DMFwere added, triethylamine (130 μL, 0.93 mmol), 50 wt % propylphosphonicanhydride solution in ethylacetate (T3P®) (0.3 mL, 0.48 mmol) andcompound 7d (160 mg, 0.37 mmol). The reaction mixture was stirred for 2hours followed by removal of the solvent under vacuum. The residue wasthen dissolved in ethylacetate and washed thrice with water and brine.The ethylacetate was then removed under vacuum to obtain the crudeintermediate bromo compound (95 mg) which was dissolved in anhydrous DMFand to it were added, triethylamine (33 μL, 0.24 mmol) and sodium azide(26 mg, 0.4 mmol). The reaction mixture was then heated at 60° C.,followed by removal of the solvent under vacuum to obtain the residuewhich was purified using column chromatography to obtain the compound 18(55 mg, 34%).¹H NMR (500 MHz, MeOD) δ 7.82 (d, J=8.3 Hz, 1H), 7.66 (d,J=8.4 Hz, 1H), 7.43 (dd, J=11.3, 4.1 Hz, 1H), 7.28 (d, J=8.1 Hz, 2H),7.12 (t, J=7.7 Hz, 1H), 7.03 (d, J=8.1 Hz, 2H), 5.87 (s, 2H), 4.37 (s,2H), 3.89 (s, 2H), 3.00-2.92 (m, 2H), 1.79 (dt, J=15.4, 7.6 Hz, 2H),1.44 (dd, J=15.0, 7.5 Hz, 2H), 0.93 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz,MeOD) δ 170.15, 156.38, 152.54, 144.50, 139.57, 136.25, 135.70, 129.50,128.68, 126.92, 125.92, 123.60, 121.68, 115.71, 52.96, 49.62, 43.62,30.88, 27.84, 23.43, 14.09. MS (ESI) calculated for C₂₄H₂₆N₈O, m/z442.2230, found 443.2345 (M+H)+.

Synthesis of Compound 19:N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pent-4-ynamide

To a solution of pentynoic acid (13 mg, 0.13 mmol) in anhydrous DMF,were added triethylamine (35 mg, 0.35 mmol), HBTU (53 mg, 0.14 mmol),and 7d (50 mg, 0.116 mol). The reaction mixture was stirred for 1 hour,followed by removal of the solvent under vacuum. The residue was thendissolved in ethylacetate and washed with water, brine, dried usingsodium sulfate and concentrated under vacuum to obtain the residue whichwas purified using column chromatography (6% MeOH/dichloromethane) toobtain compound 19 (37 mg, 73%). ¹H NMR (500 MHz, MeOD) δ 7.96 (dd,J=8.4, 0.8 Hz, 1H), 7.73 (dd, J=8.3, 0.8 Hz, 1H), 7.63 (ddd, J=8.4, 7.3,1.2 Hz, 1H), 7.36 (ddd, J=8.4, 7.3, 1.2 Hz, 1H), 7.30 (d, J=8.3 Hz, 2H),7.05 (d, J=8.3 Hz, 2H), 5.92 (s, 2H), 4.35 (s, 2H), 3.02-2.97 (m, 2H),2.49-2.43 (m, 2H), 2.43-2.38 (m, 2H), 2.23 (t, J=2.6 Hz, 1H), 1.85 (dt,J=21.1, 7.6 Hz, 2H), 1.46 (dd, J=15.0, 7.5 Hz, 2H), 0.95 (t, J=7.4 Hz,3H). ¹³C NMR (126 MHz, MeOD) δ 174.12, 159.09, 150.66, 140.32, 137.69,135.81, 135.32, 131.00, 129.65, 127.12, 126.92, 126.50, 126.47, 126.03,123.07, 119.94, 118.82, 114.41, 112.00, 83.65, 70.50, 49.96, 43.73,36.10, 30.48, 27.90, 23.45, 15.82, 14.23. MS (ESI) calculated forC₂₇H₂₉N₅O, m/z 439.24, found 440.25 (M+H)⁺.

Synthesis of Compound 21:N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide

To a solution of 20 (30 mg, 0.18 mmol) in anhydrous DMF, were addedtriethylamine (50 mg, 0.49 mmol), HBTU (68 mg, 0.18 mmol), a catalyticamount of DMAP and 7d (70 mg, 0.16 mmol). The reaction mixture wasstirred for 14 hours and then the solvent was removed under vacuum. Theresidue was dissolved in ethyl acetate and washed with water. The ethylacetate fraction was then dried using Na₂SO₄ and evaporated under vacuumto obtain the residue, which was purified using column chromatography(5% MeOH/dichloromethane) to obtain compound 21 (65 mg, 80%). ¹H NMR(500 MHz, MeOD) δ 7.97 (dd, J=8.4, 0.7 Hz, 1H), 7.74-7.71 (m, 1H), 7.64(ddd, J=8.4, 7.2, 1.2 Hz, 1H), 7.38 (ddd, J=8.4, 7.2, 1.2 Hz, 1H), 7.26(d, J=8.3 Hz, 2H), 7.04 (d, J=8.3 Hz, 2H), 6.73 (s, 2H), 5.93 (s, 2H),4.27 (s, 2H), 3.75 (t, J=7.0 Hz, 2H), 3.02-2.97 (m, 2H), 2.47 (t, J=7.0Hz, 2H), 1.85 (dt, J=21.1, 7.6 Hz, 2H), 1.46 (dq, J=14.8, 7.4 Hz, 2H),0.94 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 172.88, 172.10,159.09, 150.46, 140.15, 137.66, 135.44, 135.36, 135.21, 130.99, 129.71,126.83, 126.50, 125.89, 123.02, 119.59, 114.25, 49.85, 43.65, 35.59,35.40, 30.35, 27.78, 23.33, 14.12. MS (ESI) calculated for C₂₉H₃₀N₆O₃,m/z 510.24, found 511.25 (M+H)⁺.

Synthesis of Compound 23:(4-((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)carbamoyl)phenyl)boronicacid

To a solution of 22 (63 mg, 0.26 mmol) in anhydrous DMF, were addedtriethylamine (58 mg, 0.58 mmol), HBTU (97 mg, 0.26 mmol), a catalyticamount of DMAP and 7d (100 mg, 0.23 mol). The reaction mixture wasstirred for 1 hour, followed by removal of the solvent under vacuum. Theresidue was then dissolved in ethylacetate and washed with water, brine,dried using sodium sulfate and concentrated under vacuum to obtain thecrude boronic acid pinacolester derivative. This was then dissolved in asolution of 9:1 acetonitrile: 1N HCl and polymer-bound boronic acid (767mg, 1.15 mmol) was added to the solution. The reaction was stirred for12 hours. The beads were then filtered and the filtrate was concentratedunder reduced pressure to obtain the residue which was purified usingC₁₈ reverse-phase column chromatography (60% MeOH/H₂O) to obtaincompound 23 (83 mg, 71%). ¹H NMR (500 MHz, DMSO) (9.00 (t, J=6.0 Hz,1H), 8.19 (s, 1H), 7.98 (dd, J=13.6, 5.5 Hz, 1H), 7.87-7.77 (m, 4H),7.63 (d, J=8.8 Hz, 2H), 7.44-7.36 (m, 2H), 7.33-7.20 (m, 3H), 7.13 (d,J=7.6 Hz, 1H), 7.01 (t, J=6.3 Hz, 2H), 5.87 (d, J=5.6 Hz, 2H), 4.42 (d,J=5.9 Hz, 2H), 2.91 (dd, J=14.4, 6.8 Hz, 2H), 1.70 (dd, J=15.3, 7.8 Hz,2H), 1.42-1.32 (m, 2H), 0.86 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO)(166.28, 150.81, 150.78, 150.60, 138.98, 135.47, 134.71, 133.90, 127.79,127.72, 126.06, 125.47, 120.48, 114.73, 113.91, 113.77, 47.89, 42.12,40.12, 40.06, 39.97, 39.89, 39.80, 39.73, 39.64, 39.56, 39.47, 39.30,39.14, 38.97, 29.46, 26.18, 21.79, 13.66.

Synthesis of Compound 25:1-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-3-(3-((4-((3-aminonopropyl)thiourea

To solution of compound 24 (23 mg, 0.046 mmol) in pyridine was addedcompound 8 (18 mg, 0.046 mmol). The reaction mixture was then heated at45° C. for 12 hours followed by removal of the solvent under vacuum. Theresidue was then purified using column chromatography to obtain theBoc-protected intermediate, which was then dissolved in 1 mL of 4N HClsolution in dioxane and stirred for 2 hours followed by removal of thesolvent under vacuum to obtain compound 25 (32 mg, 89%). ¹H NMR (500MHz, MeOD) δ 7.96 (d, J=8.4 Hz, 1H), 7.76 (d, J=8.4 Hz, 1H), 7.64 (t,J=7.8 Hz, 1H), 7.37 (dd, J=8.3, 7.3 Hz, 1H), 7.32 (d, J=8.1 Hz, 2H),7.06 (d, J=8.0 Hz, 2H), 5.94 (s, 2H), 4.74-4.60 (m, 2H), 3.67 (s, 2H),3.12 (dd, J=16.0, 8.3 Hz, 2H), 3.09-2.96 (m, 10H), 2.08 (dd, J=9.0, 6.5Hz, 2H), 1.99-1.88 (m, 2H), 1.88-1.71 (m, 6H), 1.47 (dd, J=15.0, 7.5 Hz,2H), 0.95 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 162.69, 162.39,159.00, 150.58, 137.58, 135.39, 135.33, 130.92, 129.46, 126.81, 126.36,125.89, 122.93, 119.59, 114.20, 49.83, 48.26, 48.01, 45.95, 45.86,41.58, 37.79, 30.34, 27.80, 27.76, 25.39, 24.36, 24.33, 24.21, 23.32,14.09. MS (ESI) calculated for C₃₃H₄₉N₉S, m/z 603.38, found 604.39(M+H)⁺.

As shown in FIGS. 3A and 3B, several imidazoquinoline derived compoundsof the present disclosure show differential TLR7/TLR8 activities. Theretention of TLR7-agonistic activity with attenuation of TLR8-agonisticactivity may result in immune modifiers with low proinflammatoryproperties.

Example 3 Fluorescent Imidazoquinoline Derived Compounds

Compound 7d served as a convenient precursor for the covalent attachmentof fluorophores without significant loss of activity. Fluorescencemicroscopy experiments show that the fluorescent analogues areinternalized and distributed in the endosomal compartment. Flowcytometry experiments using whole human blood show differentialpartitioning into B, T, and natural killer (NK) lymphocytic subsets,which correlate with the degree of activation in these subsets. Thesefluorescently-labeled imidazoquinolines will likely be useful inexamining in detail the trafficking of TLR7 in immunological synapses.

The free primary amine on the N¹ substituent of 7d was covalentlycoupled directly to commercially-available fluorescein isothiocyanateand rhodamine B isothiocyanate (Scheme 6). Conversely, the amine on 7dwas converted first to the isothiocyanate 8, allowing the subsequentcoupling of amine-bearing fluorophores, such as the bora-diazaindacenedye, BODIPY-TR-cadaverine (Scheme 6, overleaf).

The syntheses of fluorescent imidazoquinoline analogues that retainTLR7-agonistic activity are expected to be useful probes in examiningthe anatomical basis of their potential immunostimulatory and adjuvanticproperties. All three fluorescent conjugates retain TLR7-agonisticactivity, although their potencies are slightly attenuated relative tothe parent compound, 7d (FIG. 4).

Synthesis of Compound 26: 2-(3-(4-((4-Amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)thioureido)-6-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoicacid

To a solution of fluorescein isothiocyanate (17 mg, 0.043 mmol) inanhydrous MeOH, were added triethylamine (13 mg, 0.13 mmol) and 7d (20mg, 0.043 mmol). The reaction mixture was then heated at 45° C. for 18hours and then the solvent was removed under vacuum. The residue wasthen purified using column chromatography (22% MeOH/dichloromethane) toobtain compound 26 (3 mg, 10%). ¹H NMR (500 MHz, DMSO) δ 10.13 (s, 3H),8.44 (s, 1H), 8.21 (s, 1H), 7.88-7.67 (m, 3H), 7.62-7.53 (m, 1H), 7.33(t, J=8.1 Hz, 2H), 7.26-7.13 (m, 2H), 7.07-6.94 (m, 3H), 6.67 (d, J=2.1Hz, 2H), 6.57 (tt, J=5.4, 4.0 Hz, 5H), 5.87 (s, 2H), 4.74 (s, 2H),2.97-2.84 (m, 2H), 1.77-1.67 (m, 2H), 1.45-1.33 (m, 2H), 0.91-0.85 (m,3H). MS (ESI) calculated for C₄₃H₃₆N₆O₅S, m/z 748.25, found 749.26(M+H)⁺.

Synthesis of Compound 27:N-(9-(4-(3-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)thioureido)-2-carboxyphenyl)-6-(diethylamino)-3H-xanthen-3-ylidene)-N-ethylethanaminium

To a solution of rhodamine B isothiocyanate (50 mg, 0.12 mmol) inanhydrous dichloromethane, were added triethylamine (47 mg, 0.47 mmol)and 7d (64 mg, 0.12 mmol). The reaction mixture was then stirred for 14hours and then the solvent was removed under vacuum. The residue wasthen purified using column chromatography (50% MeOH/dichloromethane) toobtain compound 27 (16 mg, 16%). ¹H NMR (500 MHz, DMSO) b 10.06 (s, 1H),8.49 (s, 2H), 7.84 (d, J=8.4 Hz, 1H), 7.81-7.73 (m, 2H), 7.56 (dd,J=8.4, 1.0 Hz, 2H), 7.33-7.29 (m, 1H), 7.24 (d, J=8.1 Hz, 2H), 7.03-6.95(m, 3H), 6.51 (dd, J=12.7, 8.4 Hz, 4H), 6.46-6.41 (m, 4H), 5.82 (s, 2H),4.63 (s, 2H), 3.36 (dd, J=11.9, 4.8 Hz, 8H), 2.92-2.83 (m, 2H), 1.70(dt, J=15.3, 7.6 Hz, 2H), 1.36 (dq, J=14.7, 7.4 Hz, 2H), 1.10 (t, J=7.0Hz, 12H), 0.85 (t, J=7.4 Hz, 3H). MS (ESI) calculated for C₂₂H₂₃N₃, m/z859.41, found 859.41 (M)⁺.

Synthesis of Compound 28: BODIPY®-TR Cadaverine Conjugated to Compound 8

To a solution of BODIPY® TR cadaverine[5-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)amino)pentylamine]hydrochloride (Invitrogen, Inc., 10 mg, 0.02 mmol) in anhydrouspyridine, was added 8 (11 mg, 0.03 mmol). The reaction mixture was thenheated at 45° C. for 18 hours and the solvent was then removed undervacuum. The residue was purified using column chromatography (8%MeOH/dichloromethane) to obtain compound 28 (2.34 mg, 15%). ¹H NMR (400MHz, MeOD) δ 7.99-7.93 (m, 3H), 7.82 (d, J=8.3 Hz, 1H), 7.66 (d, J=8.4Hz, 1H), 7.56 (t, J=6.7 Hz, 2H), 7.42 (s, 1H), 7.26 (t, J=7.3 Hz, 3H),7.17 (dd, J=8.5, 4.3 Hz, 2H), 7.06 (dd, J=8.9, 2.6 Hz, 3H), 6.95 (d,J=8.0 Hz, 2H), 6.83 (d, J=4.3 Hz, 1H), 6.74 (d, J=4.1 Hz, 1H), 5.73 (s,2H), 4.57 (s, 2H), 3.22 (ddd, J=25.7, 16.1, 9.0 Hz, 4H), 2.88-2.83 (m,2H), 1.76 (dt, J=15.3, 7.6 Hz, 3H), 1.51 (dt, J=18.8, 9.6 Hz, 4H), 1.39(dd, J=15.1, 7.5 Hz, 2H), 1.36-1.24 (m, 3H), 0.90 (t, J=7.4 Hz, 3H). MS(ESI) calculated for C₄₉H₅₁BF₂N₉O₂S₂, m/z 910.37, found 910.37 (M⁺).

Fluorescence Microscopy:

Murine macrophage J774.A1 cells were grown to confluency inoptical-grade flat-bottomed 96 well plates as described earlier.^(25,26)The cells were then exposed to graded concentrations of thefluorescently labeled compounds for 4h at 37° C. Intravitalepifluorescence and phase contrast images were obtained directly fromthe plated cells using an inverted Olympus IX-71 microscope equippedwith long working-distance air objectives and temperature-controlledstage, using appropriate filter sets for the various fluorescentanalogues. Images were processed on Image-J software.

Flow-Cytometric Immunostimulation Experiments:

Methodology for flow-cytometirc immunostimulation is as described inKawai et al., 2007.¹ Heparin-anticoagulated whole blood samples wereobtained by venipuncture from healthy human volunteers with informedconsent and as per guidelines approved by the University of Kansas HumanSubjects Experimentation Committee. Two mL aliquots of whole human bloodsamples were stimulated with graded concentrations of 26 in a 6-wellpolystyrene plate and incubated at 37° C. in a rotary (100 rpm)incubator for 30 min. Negative (endotoxin free water) controls wereincluded in each experiment. Following incubation, 200 μL aliquots ofanticoagulated whole blood were stained with 20 μL offluorochrome-conjugated antibodies (anti-CD3-PE, and anti-CD56-APC) at37° C. in the dark for 30 min. Following staining, erythrocytes werelysed and leukocytes fixed in one step by mixing 200 μL of the samplesin 4 mL pre-warmed Whole Blood Lyse/Fix Buffer (Becton-DickinsonBiosciences, San Jose, Calif.). After washing the cells twice at 200 gfor 8 minutes in saline, the cells were transferred to a 96-well plate.Flow cytometry was performed using a BD FACSArray instrument in thetri-color mode. The primary gate for the lymphocytic population wasobtained on FSC and SSC channels (100,000 gated events). Secondarygating included natural killer lymphocytes (NK cells: CD3-CD56), nominalB lymphocytes (CD3-CD56-), and nominal T lymphocytes (CD3⁺CD56⁻).Post-acquisition analyses were performed using FlowJo v 7.0 software(Treestar, Ashland, Oreg.). Compensation for spillover was computed foreach experiment on singly-stained samples.

Incubation of murine macrophage J774.A1 cells with 27 and 28, followedby intravital epi- and confocal fluorescence microcopy showed prominentperinuclear localization, which is consistent with the expectedendosomal distribution of TLR7.²⁷ Shown in FIG. 5 is a representativeepifluorescence micrograph of J774 cells treated with 28 at 100 nMconcentration.

Earlier immunoprofiling of the TLR7-agonistic imidazoquinolines hadshown a very prominent activation of B- and NK-cells, but minimalactivation of T cells,⁸ which was believed to be potentially due todifferential uptake of the TLR7 agonist in lymphocytic subsets. Flowcytometric analysis of the FITC-labeled 26 in experiments employingwhole human blood indeed demonstrated a prominent uptake of 26 inCD3-CD56 NK and CD3-CD56-B lymphocytes as compared to CD3⁺CD56⁻ Tlymphocytes (FIG. 6).

Example 4 Self-Adjuvanting Imidazoquinoline Derived Compounds

One aspect of the present work in the area of evaluating TLR agonists asvaccine adjuvants^(8,24,28) focuses on developing self-adjuvantingvaccine constructs, e.g., antigen covalently coupled to a suitableadjuvant. The premise of covalently decorating protein antigens withpotential adjuvants offers the possibility of drastically reducingsystemic exposure of the adjuvant, and yet maintaining relatively highlocal concentrations at the site of vaccination.²⁹ Most self-adjuvantingvaccine constructs to date have utilized TLR-2 agonistic2,3-bis-(palmitoyloxy)propyl-cysteinyl peptides as the adjuvant.³⁰⁻³⁵The conjugation of the poorly soluble lipopeptide adjuvant to antigenhas limited this approach to peptide^(31-34,36) or glycopeptide³⁵antigens, since native proteins are often irrevocably denatured underthe coupling conditions employed, with potential loss of key epitopes.These limitations have recently been addressed by appending to thelipopeptide a long, water-solubilizing poly-lysine or polyethyleneglycol moiety, and terminating in a free thiol.³⁷ However, in additionto the potential problem of oxidation of lipopeptide thiol to thedisulfide, free exposed thiols in proteins are rare.³⁸ Furthermore, TLR2ligation has been associated with Th2 and Th17 responses^(39,40) whichmay, in many instances, be undesirable.

As describe above, the present disclosure provides potent TLR7 agonists,stimulating virtually all subsets of lymphocytes without inducingdominant proinflammatory cytokine responses.⁸ A TLR7/8 dual-agonisticN-(4-aminomethyl)benzyl substituted imidazoquinoline 7d served as aconvenient precursor for the syntheses of isothiocyanate and maleimideimidazoquinoline derived compounds for covalent attachment to free amineand thiol groups of peptides and proteins. Compound 7d was also amenableto direct reductive amination with maltoheptaose without significantloss of activity. Covalent conjugation of the isothiocyanate derivative8 to α-lactalbumin could be achieved under mild, non-denaturingconditions, in a controlled manner and with full preservation ofantigenicity. The self-adjuvanting α-lactalbumin construct inducedrobust, high-affinity immunoglobulin titers in murine models. Thepremise of covalently decorating protein antigens with adjuvants offersthe possibility of drastically reducing systemic exposure of theadjuvant, and yet eliciting strong, Th1-biased immune responses.

Desirous of specifically identifying chemotypes with strong Th1-biasedimmunostimulatory signatures, additional screens were implemented thatexamine the induction of Type I⁴¹⁻⁴³ and Type II^(44,45) interferons(IFN-α/β and IFN-γ, respectively), Interleukin-12 (IL-12),^(46,47) andInterleukin-18 (IL-18) using human PBMCs,⁴⁸⁻⁵⁰ all of which are stronglyassociated with dominant Th1 outcomes. These experiments enableddetermination that of all of the diverse chemotypes of the presentlydescribed and rapidly expanding libraries of TLR agonists,^(24,28,51,52)an N¹-(4-aminomethyl)benzyl substituted imidazoquinoline 7d displayed aprominent Th1 bias (FIGS. 7A and 7B).

Because the aqueous solubility of 7d and several of its congeners wereexcellent, direct covalent coupling to free amines and thiols on proteinantigens via the introduction of conventional isothiocyanate andmaleimide electrophilic handles on the imidazoquinoline scaffold wasevaluated. The isothiocyanate derivative 8 (synthesis shown in Scheme 2)reacted well with a tri-glycine methyl ester model peptide, yielding theadduct 29 (Scheme 7), which was purified to homogeneity, and found to beactive (FIG. 8). Facile adduction of the maleimide derivative 21(synthesis shown in Scheme 4) with glutathione (reduced) also affordedthe 30a in near-quantitative yields (Scheme 7).

Synthesis of Compound 29: Methyl1-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)phenyl)-6,9-dioxo-3-thioxo-2,4,7,10-tetraazadodecan-12-oate

To a solution of 8 (15 mg, 0.037 mmol) in anhydrous MeOH, were addedtriethylamine (6 mg, 0.056 mmol) and methyl2-(2-(2-aminoacetamido)acetamido)acetate hydrochloride (11 mg, 0.044mmol). The reaction was heated at 45° C. for 4 hours. The solvent wasthen removed under vacuum and the residue was purified using columnchromatography (14% MeOH/dichloromethane) to obtain compound 29 (5 mg,22%). ¹H NMR (400 MHz, DMSO) δ 8.36-8.14 (m, 3H), 7.79 (d, J=7.7 Hz,1H), 7.58 (d, J=7.5 Hz, 2H), 7.33 (dd, J=11.2, 4.1 Hz, 1H), 7.25 (d,J=8.2 Hz, 2H), 7.05 (t, J=7.1 Hz, 1H), 7.00 (d, J=8.1 Hz, 2H), 6.59 (s,2H), 5.85 (s, 2H), 4.61 (s, 2H), 4.11 (s, 2H), 3.83 (d, J=5.9 Hz, 2H),3.75 (d, J=5.9 Hz, 2H), 3.62 (s, 3H), 2.96-2.86 (m, 2H), 1.72 (dt,J=15.3, 7.6 Hz, 2H), 1.45-1.31 (m, 2H), 0.88 (t, J=7.4 Hz, 3H). MS (ESI)calculated for C₃₀H₃₆N₈O₄S, m/z 604.26, found 605.27 (M+H)⁺.

Synthesis of Compound 30b: (2R)-methyl2-amino-5-((2S)-3-(1-(3-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzylamino)-3-oxopropyl)-2,5-dioxopyrrolidin-3-ylthio)-1-(2-methoxy-2-oxoethylamino)-1-oxopropan-2-ylamino)-5-oxopentanoate

To a solution of 21 (15 mg, 0.03 mmol) in anhydrous MeOH and a few dropsof anhydrous dichloromethane, were added triethylamine (8 mg, 0.08 mmol)and glutathione reduced dimethyl ester (20 mg, 0.06 mmol).[Glutathione-reduced dimethyl ester was obtained fromglutathione-reduced by stirring in mixture of methanol and 1 mL ofHCl/dioxane solution for 30 hours, followed by removal of the solventunder vacuum]. The reaction mixture was stirred for 30 minutes, followedby removal of solvent under vacuum. The residue was then purified usingcolumn chromatography (20% MeOH/dichloromethane) to obtain compound 30b(5 mg, 60%). ¹H NMR (500 MHz, MeOD) δ 7.75 (d, J=8.3 Hz, 1H), 7.57 (d,J=8.0 Hz, 1H), 7.36 (t, J=7.3 Hz, 1H), 7.17 (d, J=8.2 Hz, 2H), 7.06 (t,J=7.7 Hz, 1H), 6.94 (d, J=8.1 Hz, 2H), 5.79 (s, 2H), 4.19 (qd, J=15.2,6.7 Hz, 2H), 3.84 (s, 2H), 3.68-3.61 (m, 5H), 3.58 (s, 3H), 3.54 (dt,J=10.7, 4.5 Hz, 1H), 3.14-2.93 (m, 3H), 2.93-2.82 (m, 2H), 2.39 (td,J=6.9, 2.4 Hz, 2H), 2.36-2.27 (m, 3H), 2.05-1.91 (m, 1H), 1.90-1.78 (m,1H), 1.75-1.66 (m, 2H), 1.40-1.30 (m, 2H), 1.21 (t, J=7.3 Hz, 2H), 0.84(t, J=7.4 Hz, 3H). MS (ESI) calculated for C₄₁H₅₁N₉O₉S, m/z 845.35,found 868.33 (M+Na⁺).

Immunoassays for Interferon (IFN)-α, IFN-γ, Interleukin (IL)-12, andIL-18.

Fresh human peripheral blood mononuclear cells (PBMC) were isolated fromhuman blood obtained by venipuncture with informed consent and as perinstitutional guidelines on Ficoll-Hypaque gradients as describedelsewhere.⁵³ Aliquots of PBMCs (10⁵ cells in 100 L/well) were stimulatedfor 12 h with graded concentrations of test compounds. Supernatants wereisolated by centrifugation, diluted 1:20, and were assayed intriplicates using a high-sensitivity analyte-specific ELISA kits (PBLInterferon Source, Piscataway, N.J. and R&D Systems, Inc., Minneapolis,Minn.).

Protein Adduction and Mass Spectrometry Experiments:

Bovine α-lactalbumin (Sigma-Aldrich Chemical Co., St. Louis, Mo., andclinical grade human serum (Talecris Biotherapeutics, Research TrianglePark, N.C.) were incubated with 8 and 21, respectively at a molar ratioof 1:5 (protein:imidazoquinoline) in aqueous carbonate buffer at pH 8.0overnight. The adducted proteins were analyzed by reverse-phaseLC-ESI-MS performed on a Shimadzu LC system (LC-10AD binary pumps,SCL-10A diode array detector) using a Zorbax 3.0 mm×150 mm 3.5 μmstable-bond C₁₈ reverse-phase column with a forty-minute binary gradient(CH₃CN/water, 0.1% HCOOH) from 5% to 95% of CH₃CN. ESI-MS data wasacquired on an Agilent LC/MSD-TOF instrument with a mass accuracy of 20ppm and a range of 100-3500 Daltons. Calibration drift was minimized ona scan-by-scan basis by using internal standards corresponding to922.0001 and 2721.0201 marker ions infused concurrently through a secondnebulizer in the ionization chamber. Deconvolution was performed usingon-board Agilent MassHunter software.

Animal Experiments:

All experiments were performed in accordance with animal care protocolsapproved by the University of Kansas IACUC Committee. Cohorts of 5outbred CF-1 mice per group were immunized on Day 0 with vehicle(control 1), 50 μg/animal of bovine α-lactalbumin alone (control 2), orα-lactalbumin covalently coupled with 5 equivalents of 8, orα-lactalbumin mixed with 5 equivalents of 7d (control 3). All antigenpreparations were in sterile, physiological saline (vehicle). A volumeof 0.2 mL was injected intramuscularly into the flank region. Animalswere boosted once on Day 14, and bled by terminal cardiac puncture(under isoflurane anesthesia) on Day 21. Sera were obtained from clottedblood by centrifugation at 3000 g×10 min, and stored at −80° C. untilassayed.

Enzyme-Linked Immunosorbent Assays (ELISA):

A precision 2000 liquid handler (Bio-Tek, Winooski, Vt.) was used forall serial dilution and reagent addition steps, and a Bio-Tek ELx405384-well plate washer was employed for plate washes; 100 mMphosphate-buffered saline (PBS) pH 7.4, containing 0.1% Tween-20 wasused as wash buffer. Nunc-Immuno MaxiSorp (384-well) plates were coatedwith 30 μL of α-lactalbumin in 100 mM carbonate buffer, pH 9.0 overnightat 4° C. After 3 washes, the plates were blocked with 3% bovine serumalbumin (in PBS, pH 7.4) for 1 h at rt. Serum samples (in quadruplicate)were serially diluted in a separate 384-well plate using the liquidhandler. After three additional washes of the assay plate, 30 μL of theserum dilutions were transferred using the liquid handler, and the plateincubated at 37° C. for 2 h. The assay plate was washed three times, and30 μL of 1:10,000 diluted appropriate anti-mouse immunoglobulin (IgG [γchain], IgM [μ chain], IgG1, IgG2a) conjugated with horseradishperoxidase was added to all wells. Following an incubation step at 37°C. for 1 h, and three washes, tetramethylbenzidine substrate was addedat concentrations recommended by vendor (Sigma). The Chromogenicreaction was terminated at 30 min by the addition of 2M H₂SO₄. Plateswere then read at 450 nm using a SpectraMax M4 device (MolecularDevices, Sunnyvale, Calif.). Data visualization and statistics(Student's T test for significance) were performed using Origin 7.0(Northampton, Mass.).

The imidazoquinolines themselves are small, non-polar, and basic, andtherefore gain access to the endolysosomal compartment in which TLR7 ispredominantly sequestered. The human embryonic kidney reporter celllines stably transfected with TLR7 (and reporter secreted alkalinephosphatase genes) that were employed in our primary screen are notprofessional phagocytic cells. There was concern if the trans-membranepermeability of the bulky, dianionic adduct 30a would be sufficient totrigger activation; thus, the conjugate of 21 with the dimethyl ester ofreduced glutathione (30b) was also tested. The adducts 29 and 30bretained activity (EC₅₀: 269 nM and 2.2 μM), while 30a was inactive(FIG. 8), indicating that trans-cellular transport of the polar adductwith two net negative charges was insufficient.

The principle of electrophile-mediated conjugation was applied to bovineα-lactalbumin as a model antigen for self-adjuvanting vaccine constructsnot only because it lent itself eminently well to rigorouscharacterization by electrospray ionization mass spectrometry (ESI-MS)methods (FIG. 9), but also because it is being evaluated as a potentialantigen for breast cancer vaccines.⁵⁴

5 equivalents of 8 were reacted with bovine α-lactalbumin in isotonicaqueous buffer at pH 8.5. Direct LC-ESI-MS evidence was obtained forcovalent adduction of 8 with the protein, indicating a remarkablybeautiful and precise Gaussian distribution of adducted species, withthe preponderant conjugate corresponding to a 1:5 molar ratio ofprotein:8 (FIG. 9).

It was of particular interest to evaluate this 8:α-lactalbumin conjugate(FIG. 9) as a self-adjuvanting subunit vaccine construct. It was ofinterest whether the conjugation procedure would preserve antigenicityof the protein, and whether the covalently-adducted construct would besuperior to a physical mixture of α-lactalbumin and 7d. Cohorts (5 pergroup) of outbred CF-1 mice were immunized with 50 μg per animal ofα-lactalbumin, or 50 μg of α-lactalbumin covalently conjugated with 5equivalents of 8, or a mixture of 50 μg of α-lactalbumin and 5equivalents of 7d. The animals were boosted once after two weeksfollowing the priming dose, and bled after an additional week.α-lactalbumin-specific IgM, IgG, as well as IgG1 and IgG2a (isotypescharacteristic of Th2 and Th1 responses,⁵⁵ respectively) were quantifiedby ELISA (FIGS. 10A-10D). As shown in FIGS. 10A-10D, dramaticenhancements in antibody titers were observed with both covalently- andnon-covalently adjuvanted protein (relative to α-lactalbumin alone).Modest, but consistent, and statistically significant differences werealso observed in titers between the covalently coupled self-adjuvantingconstruct, and mixture of antigen and adjuvant, indicating thatself-adjuvanting subunit protein vaccines may indeed be generated withfull preservation of antigenicity. Examination of the affinity ofantigen-specific IgG using conventional chaotropic ELISA^(56,57) alsoindicates higher quality IgG (FIG. 11) elicited by the self-adjuvantingconstruct.

Encouraged by these results, conjugation of 21 with human serum albumin(HSA), a 66 kDa protein with a single free thiol was also performed.Clinical grade (meant for human parenteral use, formulated with aminoacids) HSA was used, rather than the purer, ‘essentially fatty acidfree’ protein available commercially. Furthermore, HSA is a carrierprotein which binds promiscuously to a vast range of ligands includingheavy metals, bilirubin, fatty acids, etc. For these reasons, it wasanticipated that deconvoluted direct electrospray-time-of-flight mass(ESI-TOF) spectra would be polydisperse and microheterogeneous. In theHSA-alone control sample, two major peaks were found corresponding to66439.9819 and 66558.3027 Da (FIG. 12). Reaction of HSA with 21 produceda shift in one of the peaks with a Δ_(mass) of 508.814 Da, whichcorresponds to the maleimide derivative 21 within instrument error(1.424 Da at 66 KDa=21.2 parts per million). The other peak at 66558 Dahad remained unaltered upon addition of excess thiol-specific maleimideanalogue 21, suggesting that the thiol was unreactive (FIG. 12). Anexamination of the difference between the species with the free,reactive thiol (66439 Da) and the unreactive thiol (66558 Da) in thecontrol sample suggests that the ‘blocking’ group is cysteine (expectedexact mass of cysteine−1 proton [disulfide]=118.02; observedΔ_(mass)=118.321 Da).

Example 5

Self-Adjuvanting Polysaccharide Vaccines:

Aside from engineering otherwise feebly immunogenic peptide and subunitprotein vaccines for the induction of strong CTL responses, there isalso interest in polysaccharide vaccines which have proved enormouslyuseful in the prevention of infections by bacterial pathogens such as N.meningitidis and H. influenzae. ⁵⁸⁻⁶⁰ Bacterial polysaccharides, unlikeconventional protein antigens, have been considered classic Tcell-independent antigens that do not elicit cell-mediated immuneresponses but rather elicit non-anamnestic responses characterized bylow-affinity IgM and restricted classes of IgG immunoglobulins withoutthe recruitment of T cell help. Conversion to canonical, Tlymphocyte-dependent responses require their covalent coupling toimmunogenic ‘carrier proteins’ such as diphtheria toxoid.^(61,62) Thisappears not to be the case for zwitterionic polysaccharides, however,which elicit potent CD4⁺ T cell responses.^(63,64) The structuraldeterminants of T-dependent and -independent humoral responses can bereexamined, especially in light of recent findings of intrinsic TLR2activation by zwitterionic polysaccharides.^(65,66) The free primaryamine on the N¹ substituent of 7d lent itself well to direct reductiveamination with maltoheptaose, a model oligosaccharide with a reducingterminal maltose unit (31, Scheme 8) which was found to be active inTLR7 assays (EC₅₀: 528 nM; FIG. 13). This method of direct coupling of7d to oligosaccharides and polysaccharides will therefore be useful forconstructing self-adjuvanting polysaccharide vaccines (such as againstN. meningitidis Group C polysaccharide).⁶⁷

Synthesis of Compound 31

To a solution of compound 7d (8 mg, 0.019 mmol) in anhydrous DMF, wereadded 3-4 drops of acetic acid, maltoheptaose (20 mg, 0.018 mmol) andmacroporous polystyrene-bound cyanoborohydride (15 mg, 0.033 mmol). Thereaction mixture was heated at 50° C. for 24 hours. The solution wasfiltered to remove the solid resin and the filtrate was evaporated undervacuum to obtain the residue which was purified using C₁₈ reverse-phasecolumn chromatography (40% MeOH/H₂O) to obtain compound 31 (12 mg, 45%).MS (ESI) calculated for C₆₄H₉₇N₅O₃₅, m/z 1495.60, found 1518.59 (M+Na⁺)and 759.83 (M+H+Na)²⁺.

Thus, 7d, with its free amine group, can be conveniently exploited inconstructing covalent conjugates with peptides, proteins, as well aspolysaccharides with preservation of immunostimulatory activity.

Example 6

Tissue-Specific Imidazoquinoline Compounds

Tissue-specific activity of 7d, can be enhanced by appending specificmoieties that undergo selective uptake by particular cell-types. Forinstance, a selective delivery of drugs to liver can be obtained byconjugation with galactosyl-terminating molecules.⁶⁸ Selective targetingof tumors can be achieved by conjugation of drugs to vitamins such asfolic acid.⁶⁹⁻⁷¹ Accordingly, the free amine group of 7d was coupled togalactose (Scheme 9) as well as a range of vitamins such as folic acid(vitamin Be), biotin (vitamin B₇), and Pyridoxal (vitamin B₆) as shownin Schemes 10-12. Several analogues retain TLR7-stimulatory activity(FIGS. 14A (TLR7 activity) and 14B (TLR8 activity)).

Synthesis of Compound 33:N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-N-((2R,3S,4R)-2,3,4,5,6-pentahydroxyhexyl)acetamide

To a solution of 7d (30 mg, 0.076 mmol) in anhydrous DMF, were addedgalactose 32 (13 mg, 0.072 mmol), 4 drops of acetic acid and MP-CNBH₃(50 mg, 0.114 mmol). The reaction mixture was heated at 50° C. for 2hours and then filtered to obtain the filtrate. The filtrate was thenconcentrated under vacuum to obtain the residue to which anhydrous DMFwas added, followed by the addition of triethylamine (10 eq.) and aceticanhydride (10 eq.) to acetylate the alcohols and the amines. The solventwas then removed and the residue was purified using columnchromatography to obtain the per-acetylated product. The O-acetyl andC4-N-acetyl groups were then removed using sodium methoxide in methanolto obtain the compound 33 (9 mg, 21%).

Synthesis of Compound 35:5-((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)amino)-2-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-5-oxopentanoicacid

To a solution of folic acid, 34 (50 mg, 0.11 mmol) in anhydrous DMSO (1mL), were added triethylamine (34 mg, 0.34 mmol), HBTU (50 mg, 0.13mmol) and 7d (36 mg, 0.08 mmol). The reaction mixture was stirred for 14hours followed by precipitating the solid using 50 mL of diethyl ether.The solid was thoroughly washed with diethyl ether 3 times and driedunder vacuum to obtain the solid which was purified using semipreparative reverse phase column chromatography to obtain the compound35 (3 mg, 5%). ¹H NMR (500 MHz, DMSO) δ 8.62 (d, J=3.9 Hz, 1H), 8.31 (d,J=6.0 Hz, 3H), 7.74-7.67 (m, 1H), 7.64 (d, J=8.7 Hz, 1H), 7.56 (d, J=8.4Hz, 2H), 7.36-7.26 (m, 2H), 7.23-7.14 (m, 2H), 7.05-6.90 (m, 4H), 6.62(dd, J=8.7, 5.4 Hz, 4H), 5.83 (d, J=10.9 Hz, 2H), 4.47 (d, J=5.8 Hz,1H), 4.38-4.29 (m, 1H), 4.25-4.15 (m, 2H), 2.92-2.86 (m, 2H), 2.27-2.18(m, 1H), 1.96 (d, J=10.0 Hz, 1H), 1.86 (s, 1H), 1.75 (s, 2H), 1.77-1.66(m, 2H), 1.40-1.34 (m, 2H), 1.23 (s, 2H), 0.85 (tt, J=4.8, 3.8 Hz, 3H).MS (ESI) calculated for C₄₁H₄₂N₁₂O₅, m/z 782.34, found 783.35 (M+H)⁺.

Synthesis of Compound 37:N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide

To a solution of biotin 36 (30 mg, 0.069 mmol) in anhydrous DMF, wereadded triethylamine (17 mg, 0.076 mmol), propylphosphonic anhydridesolution in ethylacetate (26 mg, 0.083 mmol) and 7d (30 mg, 0.069 mmol).The reaction mixture was stirred for 1 hour, followed by removal of thesolvent under vacuum. The residue was then dissolved in ethylacetate andwashed with water, brine, dried using sodium sulfate and concentratedunder vacuum to obtain the residue which was purified using columnchromatography (25% MeOH/dichloromethane) to obtain compound 36 (25 mg,62%). ¹H NMR (500 MHz, MeOD) δ 7.84 (d, J=8.3 Hz, 1H), 7.63 (d, J=8.3Hz, 1H), 7.51 (t, J=7.8 Hz, 1H), 7.24 (t, J=7.8 Hz, 1H), 7.18 (d, J=8.1Hz, 2H), 6.95 (d, J=8.0 Hz, 2H), 5.82 (s, 2H), 4.40-4.34 (m, 1H),4.24-4.18 (m, 2H), 4.15 (dd, J=7.8, 4.4 Hz, 1H), 3.56 (s, 1H), 3.13-2.98(m, 2H), 2.90 (t, J=7.6 Hz, 2H), 2.59 (t, J=13.3 Hz, 1H), 2.25 (t, J=7.4Hz, 1H), 2.12 (t, J=7.3 Hz, 1H), 1.78-1.70 (m, 2H), 1.62-1.48 (m, 5H),1.36 (dd, J=7.5, 5.6 Hz, 2H), 0.85 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz,MeOD) δ 175.97, 166.10, 158.69, 150.75, 140.43, 137.36, 135.29, 130.65,129.49, 129.14, 126.86, 126.04, 122.82, 120.51, 114.44, 63.39, 61.61,57.03, 56.98, 52.03, 49.81, 49.64, 49.52, 49.47, 49.35, 49.30, 49.18,49.01, 48.84, 48.67, 48.50, 43.54, 41.05, 36.70, 34.55, 30.77, 30.45,29.75, 29.72, 29.50, 29.47, 27.79, 26.86, 25.92, 23.35, 14.11. MS (ESI)calculated for C₃₂H₃₉N₇O₂S, m/z 585.28, found 586.29 (M+H)⁺.

Synthesis of Compound 39:4-(((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)amino)methyl)-5-(hydroxymethyl)-2-methylpyridin-3-ol

To a solution of compound 7d (50 mg, 0.116 mmol) in anhydrous methanol,were added pyridoxal hydrochloride (24 mg, 0.116 mmol) and sodiumtriacetoxyborohydride (77 mg, 0.35 mmol). The reaction mixture wasstirred for 2 hours followed by removal of the solvent under vacuum. Theresidue was then dissolved in ethylacetate and washed with saturatedsodium bicarbonate solution, water, brine, dried using sodium sulfateand concentrated under vacuum to obtain the residue which was purifiedusing column chromatography (20% MeOH/dichloromethane) to obtaincompound 39 (7 mg, 12%). ¹H NMR (500 MHz, DMSO) δ 7.79 (d, J=7.5 Hz,1H), 7.75 (s, 1H), 7.57 (dd, J=8.3, 0.9 Hz, 1H), 7.32 (s, 1H), 7.28 (d,J=8.2 Hz, 2H), 7.06-7.00 (m, 3H), 6.54 (s, 2H), 5.86 (s, 2H), 5.12-4.93(m, 1H), 4.33 (s, 2H), 3.92 (s, 2H), 3.64 (s, 2H), 2.93-2.88 (m, 2H),2.25 (s, 3H), 1.70 (d, J=7.6 Hz, 2H), 1.38 (d, J=7.5 Hz, 2H), 0.87 (t,J=7.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO) b 153.54, 152.49, 151.69, 145.14,144.78, 137.96, 137.85, 135.58, 132.81, 132.71, 128.80, 127.36, 126.42,126.27, 126.08, 125.57, 120.83, 120.04, 114.53, 99.49, 58.93, 51.22,47.80, 45.97, 29.58, 26.20, 21.82, 18.69, 13.67. MS (ESI) calculated forC₃₀H₃₄N₆O₂, m/z 510.27, found 511.28 (M+H)⁺.

Example 7 Imidazoquinoline Dendrimers

Being small, the imidazoquinolines may diffuse out quickly from theinjection site. A dendrimeric molecule bearing three (41) or six unitsof 7d (43) were synthesized as shown in Schemes 13 and 14. In the caseof 43, a loss of TLR8-stimulatory activity occurred (FIG. 15B), whileretaining in large measure the TLR7-agonistic activity of its parentmonomer (FIG. 15A). This is reflected by an absence of proinflammatorycytokine induction in human PBMCs as shown in FIGS. 16A-16E, whichillustrates selective loss of TLR8-associated cytokine induction by thedendrimer 43. There was also dissociation between TLR7-driven IFN-α andTLR8-driven IFN-γ induction (FIGS. 17A and 17B, respectively). Based oncurrent paradigms in innate immunity, it could be predicted that thedendrimer, by virtue of being a pure TLR7 agonist and consequentlyinducing IFN-α, and not proinflammatory cytokines, would manifest inlower reactogenicity (local and/or systemic inflammation). The dendrimerbehaved better than 7d in rabbit immunization model using bovineα-lactalbumin as a model subunit vaccine (FIG. 18).

Synthesis of Compound 41:1,1′,1″-(nitrilotris(ethane-2,1-diyl))tris(3-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)thiourea)

To a solution of compound 40 (4.2 mg, 0.29 mmol) in pyridine (1 mL) wasadded compound 8 (40 mg, 0.11 mmol). The reaction mixture was heated at45° C. for 2 hours, followed by the addition of Polystyrene bound-NH₂beads to quench the excess of compound 8. The reaction was stirred foranother 30 minutes, followed by filtration of the beads. The filtratethus obtained was concentrated under vacuum and the residue was washedseveral times with diethyl ether to afford compound 41 (22 mg, 56%). ¹HNMR (500 MHz, MeOD) δ 7.70 (d, J=7.9 Hz, 3H), 7.59 (dd, J=8.4, 0.8 Hz,3H), 7.36-7.32 (m, 3H), 7.05 (td, J=8.5, 4.4 Hz, 9H), 6.86 (d, J=6.9 Hz,6H), 5.63 (s, 6H), 4.46 (s, 6H), 3.48-3.38 (m, 6H), 3.04-2.99 (m, 2H),2.86-2.81 (m, 6H), 2.74-2.69 (m, 2H), 2.48 (s, 6H), 1.78-1.73 (m, 6H),1.38 (dd, J=15.0, 7.5 Hz, 6H), 0.89 (t, J=7.4 Hz, 9H). ¹³C NMR (126 MHz,MeOD) δ 156.82, 151.91, 136.19, 135.05, 129.43, 129.30, 126.85, 126.71,124.52, 121.92, 115.40, 67.09, 53.57, 38.74, 30.77, 30.68, 28.02, 23.54,15.75, 14.52. MS (ESI) calculated for C₇₅H₈₇N₁₉S₃, m/z 1349.65, found1350.66 (M+H)⁺ and 675.83 (M+2H)⁺².

Synthesis of Compound 42:N¹,N¹-bis(2-(di(prop-2-yn-1-yl)amino)ethyl)-N²,N²-di(prop-2-yn-1-yl)ethane-1,2-diamine

To a solution of compound 40 (299 μL, 2.0 mmol) in CH₃CN (20 mL) wasadded Et₃N (1.75 mL, 12.6 mmol). The reaction mixture was cooled to 0°C. and propargyl bromide (80% solution in toluene, 2 mL, 13.5 mmol) wasadded drop wise over a period of 10 min and the reaction mixture waskept stirring at room temperature for 6 hours. Water was added to thereaction mixture and the product was extracted in ethyl acetate. Theorganic layer was washed with water (2×20 mL), brine (2×20 mL) and driedover anhydrous sodium sulfate and evaporated. The crude residue wascolumn purified to afford compound 42 as thick liquid (433 mg, 58%). ¹HNMR (500 MHz, CDCl₃) δ 3.47 (d, J=2.4 Hz, 12H), 2.66 (s, 12H), 2.23 (t,J=2.4 Hz, 6H), 1.63 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 78.99, 73.23,52.76, 50.65, 42.71. MS (ESI) calculated for C₂₄H₃₀N₄, m/z 374.25, found375.25 (M+H)⁺.

Synthesis of Compound 43

To a stirred solution of compound 42 (5.0 mg, 0.013 mmol) and 3 (40 mg,0.091 mmol) in DMF (2 mL), were added CuSO₄.5H₂O (23 mg, 0.091 mmol, in0.5 mL water) and sodium ascorbate (36 mg, 0.18 mmol in 0.5 mL water)and the reaction mixture was stirred at room temperature for 1 h. Theclick dendrimer formed was purified by semipreparative reverse phaseHPLC to afford solid compound 43 (25 mg, 63%). MS (ESI) calculated forC₁₆₈H₁₈₆N₅₂O₆, m/z 3027.5848, found 1514.8233 (M+2H)⁺² and 1010.8771(M+3H)³⁺.

Example 8 Dimeric Imidazoquinoline Variants

The majority of TLRs signal via homo- or hetero-dimerization,⁷² and itwas therefore of interest to examine dimeric constructs with differinggeometries. The first series of dimeric imidazoquinolines were linked atthe C2 position and were synthesized via two different routes. Whereasthe hexamethylene- and decamethylene-bridged compounds 49a and 49b couldbe conveniently obtained from 48 by a direct, one-step, bis-amidationusing the corresponding dicarboxylic acid chlorides and cyclization(Scheme 15), the shorter chain analogues were not amenable to thismethod because of intramolecular cyclization, giving rise to undesiredquinolin-3-yl piperidinediones. This problem was circumvented by firstreacting glutaric or adipic anhydride with 44, which yielded themonocarboxylic imidazoquinolines 45a and 45b, respectively; theseintermediates were taken forward without purification and reacted againwith 44 to afford the C4,C4′-des-amino precursors 46a and 46b (Scheme15). Compounds 47a and 47b (with amines at C4 and C4′, respectively)were obtained by sequential N-oxidation of the quinoline nitrogen,reaction with benzoyl isocyanate to afford the C4 and C4′ N-benzoylintermediate and, finally, cleavage of the N-benzoyl group using sodiummethoxide as described by us earlier.^(24,51) Next, the dimers linkedvia the C4-NH₂ (51a-d) were synthesized by direct S_(N)Ar on 50 usingα,ω-bis-amino alkanes (Scheme 16). Similarly, dimers linked at the N¹position on the 4-aminomethylene benzyl group (52a-c) were obtainedusing appropriate dicarboxylic acid chlorides (Scheme 17).

While the dimeric imidazoquinoline derivative compounds 52a-c and 55a-care formed from dimerization of compounds of Formulas I and II of thepresent disclosure, the dimeric imidazoquinoline derivative compounds47a-b and 49a-b are formed from dimerization of a slightly altered basecompound. The dimeric structure of the 47 and 49 compounds can berepresented by the following structure illustrated by Formula III.

where R₁ is independently selected from the group consisting ofhydrogen, halogen (—Cl, —Br, —F), nitro (—NO₂), —NH₂, azido (—N₃),hydroxyl (—OH), —CF₃; R₃ is selected from the group consisting ofhydrogen or —(CH₂)_(x)—NH₂, and x is any integer form 1 to 10.

The dimeric imidazoquinoline derivative compounds 52a-c are formed fromdimerization of a compound of Formula I of the present disclosure,specifically compounds 52 a-c are dimers of compound 7d linked at the N¹position on the 4-aminomethylene benzyl group. The dimericimidazoquinoline derivative compounds 55a-c are formed from dimerizationof a compound of Formula II of the present disclosure and can berepresented by the general formula illustrated below.

Where R₂ is a C₂-C₅ alkyl, R₃ is selected from the group consisting ofhydrogen or —(CH₂)_(x)—NH₂, and x is any integer form 1 to 12.

To link the 55 series of dimers via the quinoline ring an additionalamine at position C8 was introduced. This was achieved via carefullycontrolled nitration of 7d using 1.2-1.3 equiv. of HNO₃, followed byN-Boc protection of the amine on the N¹ substituent, and subsequentreduction. Dimerization of the 4,8-diaminoimidazoquinoline 54 proceededsmoothly using dicarboxylic acid chlorides as described in the previousschemes (Scheme 18). It is to be noted that the mono-nitro andmono-amino precursors 53 and 54 were also N-Boc deprotected and testedfor TLR-modulatory activities (Table 1).

Synthesis of Compound 47a:2,2′-(propane-1,3-diyl)bis(1-benzyl-1H-imidazo[4,5-c]quinolin-4-amine)

To a solution of 44 (100 mg, 0.4 mmol) in anhydrous THF, were addedtriethylamine (53 mg, 0.52 mmol) and glutaric anhydride (60 mg, 0.52mmol) and the reaction vessel was heated in a microwave for 2 hours at110° C. The solvent was then removed under vacuum to obtain the crudeproduct 45a, which was then dissolved in anhydrous DMF and to thissolution, were added HBTU (167 mg, 0.44 mmol), triethylamine (53 mg,0.52 mmol), 44 (100 mg, 0.4 mmol) and a catalytic amount of DMAP. Thereaction mixture was stirred for 12 hours at 90° C. The solvent was thenremoved under vacuum and the residue was purified using columnchromatography (12% MeOH/dichloromethane) to obtain the intermediate thecompound 46a (157 mg). To a solution of 46a in solvent mixture ofMeOH:dichloromethane:chloroform (0.1:1:1), was added3-chloroperoxybenzoic acid (242 mg, 1.4 mmol) and the reaction mixturewas refluxed at 45° C. for 40 minutes. The solvent was then removed andthe residue was purified using column chromatography (35%MeOH/dichloromethane) to obtain the bis-N-oxide derivative (130 mg).bis-N-oxide derivative (110 mg, 0.19 mmol) was then dissolved inanhydrous dichloromethane, followed by the addition of benzoylisocyanate (96 mg, 0.67 mmol) and heated at 45° C. for 15 minutes. Thesolvent was then removed under vacuum and the residue was dissolved inanhydrous MeOH, followed by addition of excess of sodium methoxide andheated at 80° C. for 2 hours. The solvent was then removed under vacuumand the residue was purified using column chromatography (50%MeOH/dichloromethane) to obtain the compound 47a (25 mg, 11%). ¹H NMR(500 MHz, DMSO) b 14.30 (s, 2H), 9.48-8.30 (bs, 4H), 7.93 (d, J=8.3 Hz,2H), 7.77 (d, J=8.3 Hz, 2H), 7.65-7.60 (m, 2H), 7.38-7.34 (m, 2H), 7.26(t, J=7.6 Hz, 4H), 7.17 (t, J=7.4 Hz, 2H), 7.03 (d, J=7.4 Hz, 4H), 5.94(s, 4H), 3.16 (t, J=7.2 Hz, 4H), 2.44-2.35 (m, 2H). ¹³C NMR (126 MHz,DMSO) b 156.22, 148.86, 135.32, 135.30, 133.48, 129.51, 128.93, 127.57,125.47, 124.72, 124.54, 121.49, 118.31, 112.16, 48.35, 25.21, 24.43. MS(ESI) calculated for C₃₇H₃₂N₈, m/z 588.2750, found 589.2860 (M+H)⁺.

Compound 47b was synthesized similarly as described for compound 47a.

47b:2,2′-(butane-1,4-diyl)bis(1-benzyl-1H-imidazo[4,5-c]quinolin-4-amine)

¹H NMR (500 MHz, DMSO) b 13.86 (s, 2H), 8.88 (bs, 4H), 7.93 (d, J=8.2Hz, 2H), 7.83-7.79 (m, 2H), 7.66-7.61 (m, 2H), 7.39-7.34 (m, 2H), 7.27(t, J=7.6 Hz, 4H), 7.18 (t, J=7.4 Hz, 2H), 7.01 (d, J=7.4 Hz, 4H), 5.94(s, 4H), 2.99 (s, 4H), 1.83 (s, 4H). ¹³C NMR (126 MHz, DMSO) δ 156.55,148.75, 135.38, 133.62, 129.50, 128.95, 127.60, 125.42, 124.74, 124.54,121.54, 118.48, 112.28, 48.34, 26.49, 26.14. MS (ESI) calculated forC₃₈H₃₄N₈, m/z 602.2906, found 603.3272 (M+H)⁺ and 302.1705 (M+2H)²⁺.

Synthesis of Compound 49a:2,2′-(hexane-1,6-diyl)bis(1-benzyl-1H-imidazo[4,5-c]quinolin-4-amine)

To a solution of 48 (60 mg, 0.21 mmol) in anhydrous THF, were addedtriethylamine (54 mg, 0.53 mmol), and suberoyl chloride (23 mg, 0.11mmol) and the reaction mixture was stirred for 6 hours. The solvent wasthen removed under vacuum and the residue was dissolved in EtOAc andwashed with water/brine. The EtOAc fraction was then dried using sodiumsulfate and evaporated under vacuum to obtain the intermediate amidecompound, which was then dissolved in 1 mL solution of 2M ammonia inMeOH and heated at 150° C. for 15 hours. The solvent was then removedunder vacuum and the residue was purified using column chromatography(20% MeOH/dichloromethane) to obtain the compound 49a (8 mg, 12%). ¹HNMR (500 MHz, MeOD) δ 7.96 (d, J=8.3 Hz, 2H), 7.77 (d, J=8.4 Hz, 2H),7.65 (dd, J=11.5, 4.2 Hz, 2H), 7.39 (t, J=7.8 Hz, 2H), 7.31 (t, J=7.4Hz, 4H), 7.25 (t, J=7.3 Hz, 2H), 7.06 (d, J=7.4 Hz, 4H), 5.93 (s, 4H),2.97 (t, J=7.5 Hz, 4H), 1.85 (d, J=7.0 Hz, 4H), 1.43 (s, 4H). ¹³C NMR(126 MHz, MeOD) δ 158.93, 137.73, 136.20, 135.30, 131.11, 130.47,129.31, 126.65, 126.57, 125.81, 122.94, 119.65, 114.20, 50.13, 29.70,27.92. MS (ESI) calculated for C₄₀H₃₈N₈, m/z 630.3219, found 631.3415(M+H)⁺.

Compound 49b was synthesized similarly as described for compound 49a.

49b:2,2′-(decane-1,10-diyl)bis(1-benzyl-1H-imidazo[4,5-c]quinolin-4-amine)

¹H NMR (500 MHz, MeOD) δ 7.86 (d, J=8.3 Hz, 2H), 7.69 (d, J=8.4 Hz, 2H),7.51 (t, J=7.7 Hz, 2H), 7.32 (t, J=7.3 Hz, 4H), 7.28 (d, J=7.2 Hz, 2H),7.23 (t, J=7.7 Hz, 2H), 7.06 (d, J=7.4 Hz, 4H), 5.88 (s, 4H), 2.96 (t,J=7.6 Hz, 4H), 1.79 (dt, J=15.3, 7.7 Hz, 4H), 1.37 (dd, J=14.9, 7.4 Hz,4H), 1.32-1.24 (m, J=11.6 Hz, 4H), 1.23 (d, J=10.1 Hz, 4H). ¹³C NMR (126MHz, MeOD) δ 157.44, 151.60, 136.61, 130.41, 129.74, 129.22, 126.69,126.46, 124.90, 123.38, 122.15, 115.12, 50.05, 30.32, 30.25, 30.17,28.49, 28.17. MS (ESI) calculated for C₄₄H₄₆N₈, m/z 686.3845, found687.3749 (M+H)⁺ and 344.1949 (M+2H)²⁺.

Synthesis of Compound 51b:N¹,N-bis(1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-yl)octane-1,8-diamine

To a solution of 50 (50 mg, 0.14 mmol) in 1 mL of anhydrous MeOH, wasadded 1,8-diaminooctane (10 mg, 0.07 mmol) and the reaction mixture washeated at 140° C. for 4 hours.

The solvent was then removed under vacuum and the residue was purifiedusing column chromatography (8% MeOH/dichloromethane) to obtain thecompound 51b (12 mg, 22%). ¹H NMR (400 MHz, CDCl₃) δ 7.87 (d, J=8.3 Hz,2H), 7.68 (d, J=8.2 Hz, 2H), 7.41 (t, J=7.7 Hz, 2H), 7.36-7.26 (m, 6H),7.09-7.01 (m, 6H), 5.78 (s, 2H), 5.72 (s, 4H), 3.77 (dd, J=12.8, 6.6 Hz,4H), 2.92-2.83 (m, 4H), 1.87-1.74 (m, 8H), 1.58-1.50 (m, 4H), 1.45 (dt,J=15.1, 7.5 Hz, 8H), 0.93 (t, J=7.4 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ153.34, 150.78, 145.51, 135.62, 129.20, 127.93, 127.41, 127.07, 126.68,125.59, 121.34, 119.52, 114.87, 48.81, 40.76, 30.18, 29.98, 29.50,27.21, 22.56, 13.77. MS (ESI) calculated for C₅₀H₅₈N₈, m/z 770.4784,found 771.4963 (M+H)⁺ and 386.2570 (M+2H)²⁺.

Compounds 51a, 51c and 51d were synthesized similarly as described forcompound 51b.

51a:N¹,N⁴-bis(1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-yl)butane-1,4-diamine

¹H NMR (500 MHz, CDCl₃) (7.86 (d, J=8.2 Hz, 2H), 7.65 (dd, J=8.2, 1.0Hz, 2H), 7.41-7.35 (m, 2H), 7.35-7.24 (m, 6H), 7.09-6.99 (m, 6H), 5.86(s, 2H), 5.69 (s, 4H), 3.87 (s, 4H), 2.88-2.81 (m, 4H), 2.00 (s, 4H),1.76 (ddd, J=13.0, 9.0, 7.7 Hz, 4H), 1.46-1.37 (m, 4H), 0.90 (t, J=7.4Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 151.27, 148.66, 143.44, 133.52,130.85, 128.77, 127.28, 127.13, 125.85, 125.38, 124.95, 124.60, 123.50,123.40, 119.29, 117.45, 112.81, 46.71, 38.40, 28.01, 25.51, 25.10,20.47, 11.68. MS (ESI) calculated for C₄₆H₅₀N₈, m/z 714.4158, found715.4333 (M+H)⁺ and 358.2263 (M+2H)²⁺.

51c:N¹,N¹⁰-bis(1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-yl)decane-1,10-diamine

¹H NMR (500 MHz, CDCl₃) δ 7.84 (d, J=8.2 Hz, 2H), 7.66 (dd, J=8.2, 1.0Hz, 2H), 7.40-7.36 (m, 2H), 7.33-7.26 (m, 6H), 7.07-7.01 (m, 6H), 5.73(s, 2H), 5.70 (s, 4H), 3.74 (dd, J=12.7, 6.5 Hz, 4H), 2.88-2.83 (m, 4H),1.80-1.72 (m, 8H), 1.53-1.24 (m, 16H), 0.91 (t, J=7.4 Hz, 6H). ¹³C NMR(126 MHz, CDCl₃) δ 151.76, 149.23, 143.97, 134.05, 131.32, 127.81,127.63, 126.36, 125.85, 125.50, 125.10, 124.01, 123.84, 119.75, 117.95,113.30, 47.23, 39.17, 28.62, 28.39, 28.06, 27.95, 25.65, 20.98, 12.20.MS (ESI) calculated for C₅₂H₆₂N₈, m/z 798.5097, found 799.5416 (M+H)⁺and 400.2799 (M+2H)²⁺.

51d:N¹,N¹²-bis(1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-yl)dodecane-1,12-diamine

¹H NMR (500 MHz, CDCl₃) δ 7.84 (d, J=8.2 Hz, 2H), 7.65 (dd, J=8.2, 1.0Hz, 2H), 7.38 (ddd, J=8.3, 7.1, 1.3 Hz, 2H), 7.34-7.24 (m, 6H),7.06-7.00 (m, 6H), 5.74 (s, 2H), 5.69 (s, 4H), 3.74 (dd, J=12.6, 6.5 Hz,4H), 2.86 (dd, J=17.4, 9.6 Hz, 4H), 1.83-1.69 (m, 8H), 1.56-1.30 (m,20H), 0.91 (t, J=7.4 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 151.81, 149.27,144.02, 134.08, 131.36, 127.68, 126.41, 125.87, 125.53, 125.16, 124.05,119.81, 118.00, 113.34, 47.27, 39.25, 28.65, 28.44, 28.15, 28.13, 28.01,25.70, 25.68, 21.03, 12.24. MS (ESI) calculated for C₅₄H₆₆N₈, m/z826.5410, found 827.5796 (M+H)⁺ and 414.2977 (M+2H)²⁺.

Synthesis of Compound 52b:N¹,N-bis(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)octanediamide

To a solution of 7d (25 mg, 0.058 mmol) in anhydrous THF, were addedtriethylamine (15 mg, 0.15 mmol) and suberoyl chloride (6 mg, 0.029mmol). The reaction mixture was stirred for 1 hour and then the solventwas removed under vacuum. The residue was then purified using columnchromatography (30% MeOH/dichloromethane) to obtain the compound 52b (8mg, 32%)¹H NMR (500 MHz, MeOD) δ 7.65 (dd, J=8.3, 0.9 Hz, 2H), 7.55-7.51(m, 2H), 7.27 (ddd, J=8.3, 7.1, 1.2 Hz, 2H), 7.12 (d, J=8.2 Hz, 4H),6.95 (ddd, J=8.2, 7.2, 1.1 Hz, 2H), 6.88 (d, J=8.2 Hz, 4H), 5.69 (s,4H), 4.18 (s, 4H), 2.84-2.78 (m, 4H), 2.03 (t, J=7.5 Hz, 4H), 1.64 (dt,J=15.4, 7.6 Hz, 4H), 1.47-1.37 (m, 4H), 1.30 (dq, J=14.8, 7.4 Hz, 4H),1.16-1.10 (m, 4H), 0.80 (t, J=7.4 Hz, 6H). ¹³C NMR (126 MHz, MeOD) δ176.03, 156.18, 152.62, 144.89, 140.08, 136.09, 135.55, 129.44, 128.54,126.95, 126.86, 126.21, 123.42, 121.58, 115.75, 49.58, 43.58, 36.85,30.85, 29.75, 27.82, 26.74, 23.43, 14.11. MS (ESI) calculated forC₅₂H₆₀N₁₀O₂, m/z 856.4901, found 879.4711 (M+Na⁺) and 429.2430 (M+2H)²⁺.

Compounds 52a and 52c were synthesized similarly as described forcompound 52b.

52a:N¹,N⁶-bis(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)adipamide

¹H NMR (400 MHz, MeOD) δ 7.85 (d, J=7.6 Hz, 2H), 7.68 (d, J=7.8 Hz, 2H),7.52-7.45 (m, 2H), 7.26 (d, J=8.2 Hz, 4H), 7.23-7.16 (m, 2H), 7.02 (d,J=8.2 Hz, 4H), 5.85 (s, 4H), 4.30 (s, 4H), 2.99-2.93 (m, 4H), 2.19 (t,J=6.0 Hz, 4H), 1.80 (dd, J=15.3, 7.7 Hz, 4H), 1.58 (t, J=3.1 Hz, 4H),1.45 (dd, J=15.0, 7.5 Hz, 4H), 0.94 (t, J=7.4 Hz, 6H). ¹³C NMR (101 MHz,MeOD) δ 174.28, 156.00, 150.28, 138.78, 135.01, 134.27, 128.13, 128.05,125.44, 123.28, 121.97, 120.73, 113.67, 48.28, 42.15, 35.17, 29.21,26.38, 25.03, 21.96, 12.68. MS (ESI) calculated for C₅₀H₅₆N₁₀O₂, m/z828.4588, found 829.4440 (M+H)⁺ and 415.2244 (M+2H)²⁺.

52c:N¹,N¹²-bis(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)dodecanediamide

¹H NMR (500 MHz, MeOD) δ 7.69 (dd, J=8.3, 0.8 Hz, 2H), 7.54 (dd, J=8.4,0.7 Hz, 2H), 7.31 (ddd, J=8.4, 7.2, 1.2 Hz, 2H), 7.14 (d, J=8.2 Hz, 4H),7.00 (ddd, J=8.2, 7.2, 1.1 Hz, 2H), 6.90 (d, J=8.2 Hz, 4H), 5.73 (s,4H), 4.20 (s, 4H), 2.85-2.81 (m, 4H), 2.06 (t, J=7.4 Hz, 4H), 1.66 (dt,J=15.4, 7.6 Hz, 4H), 1.44 (dt, J=14.4, 7.3 Hz, 4H), 1.31 (dq, J=14.8,7.4 Hz, 4H), 1.10 (dd, J=29.4, 26.2 Hz, 12H), 0.81 (t, J=7.4 Hz, 6H).¹³C NMR (126 MHz, MeOD) δ 176.17, 156.64, 152.26, 143.34, 140.20,135.93, 135.90, 129.44, 128.92, 126.85, 126.77, 125.13, 123.90, 121.81,115.51, 49.64, 43.55, 36.99, 30.78, 30.36, 30.23, 30.11, 27.82, 26.96,23.41, 14.11. MS (ESI) calculated for C₅₆H₆₈N₁₀O₀₂, m/z 912.5527, found913.5886 (M+H)⁺ and 457.2974 (M+2H)²⁺.

Synthesis of Compound 53: tert-butyl4-((4-amino-2-butyl-8-nitro-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzylcarbamate

To a solution of 7d (500 mg, 1.16 mmol) in H₂SO₄, was added HNO₃ (95 mg,1.511 mmol). The reaction mixture was stirred for 12 hours, followed byneutralization of sulfuric acid by slow addition of sodium carbonatesolution. EtOAc was added to this solution to extract the compound,followed by washing with water/brine. The EtOAc fraction was then driedusing sodium sulfate and evaporated under vacuum to obtain the residue.The residue as dissolved in MeOH and di-tert-butyl dicarbonate was addedto it. The reaction was stirred for 30 minutes followed by removal ofthe solvent under vacuum to obtain the residue, which was purified usingcolumn chromatography (7% MeOH/dichloromethane) to obtain the compound53 (200 mg, 34%).¹H NMR (400 MHz, CDCl₃) δ 8.67 (d, J=2.5 Hz, 1H),8.24-8.18 (m, 1H), 7.76 (d, J=9.2 Hz, 1H), 7.28 (d, J=7.0 Hz, 2H), 7.08(d, J=8.1 Hz, 2H), 5.95 (s, 2H), 5.76 (s, 2H), 4.87 (s, 1H), 4.29 (d,J=5.5 Hz, 2H), 3.05-2.95 (m, 2H), 1.88 (dt, J=15.5, 7.6 Hz, 2H), 1.51(dd, J=14.9, 7.3 Hz, 2H), 1.45 (s, 9H), 0.99 (t, J=7.4 Hz, 3H). ¹³C NMR(101 MHz, CDCl₃) δ 155.18, 153.33, 148.68, 141.64, 139.53, 133.91,133.29, 128.41, 127.42, 127.25, 125.92, 121.27, 117.28, 113.88, 48.81,44.09, 30.00, 28.35, 27.20, 22.54, 13.78. MS (ESI) calculated forC₂₇H₃₂N₆O₄, m/z 504.2485, found 505.2541 (M+H)⁺.

Synthesis of Compound 53a: 1-(4-(aminomethyl)benzyl)-2-butyl-8-nitro-1H-imidazo[4,5-c]quinolin-4-amine

Compound 53 (10 mg, 0.02 mmol) was dissolved in 1 mL solution ofHCl/dioxane and stirred for 12 hours. The solvent was then removed undervacuum and the residue was washed with diethyl ether to afford thecompound 53a in quantitative yields. ¹H NMR (400 MHz, MeOD) b 8.77 (d,J=1.7 Hz, 1H), 8.47-8.41 (m, 1H), 7.98 (d, J=9.1 Hz, 1H), 7.54 (d, J=7.7Hz, 2H), 7.30 (d, J=7.7 Hz, 2H), 6.09 (s, 2H), 4.11 (s, 2H), 3.12 (t,J=7.5 Hz, 2H), 1.98-1.88 (m, 2H), 1.59-1.49 (m, 2H), 1.00 (t, J=7.3 Hz,3H). ¹³C NMR (101 MHz, MeOD) δ 158.58, 150.14, 143.93, 137.46, 135.58,135.36, 133.41, 129.86, 126.29, 125.89, 123.43, 119.40, 117.94, 112.44,48.52, 42.36, 29.05, 26.44, 21.94, 12.71. MS (ESI) calculated forC₂₂H₂₄N₆O₂, m/z 404.1961, found 405.1993 (M+H)⁺.

Synthesis of Compound 54: tert-butyl4-((4,8-diamino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzylcarbamate

To a solution of 53 (190 mg, 0.377 mmol) in anhydrous MeOH, were added acatalytic amount of Pd/C and the reaction mixture was subjected tohydrogenation at 60 psi hydrogen pressure for 4 hours. The reactionmixture was then filtered through celite and the filtrate was evaporatedunder vacuum to obtain the compound 54 (160 mg, 90%).¹H NMR (400 MHz,MeOD) δ 7.49 (d, J=8.8 Hz, 1H), 7.25 (d, J=8.1 Hz, 2H), 7.14 (d, J=2.3Hz, 1H), 7.02 (d, J=8.1 Hz, 2H), 6.97 (dd, J=8.9, 2.4 Hz, 1H), 5.75 (s,2H), 4.19 (s, 2H), 2.92-2.86 (m, 2H), 1.73 (dt, J=15.4, 7.6 Hz, 2H),1.43 (s, 9H), 0.92 (t, J=7.4 Hz, 3H). ¹³C NMR (101 MHz, MeOD) b 154.56,148.78, 142.89, 139.42, 136.71, 134.64, 133.60, 127.53, 125.80, 125.57,118.16, 115.16, 103.42, 78.81, 43.18, 29.41, 27.33, 26.41, 22.01, 12.67.MS (ESI) calculated for C₂₇H₃₄N₆O₂, m/z 474.2743, found 475.2733 (M+H)⁺.

Synthesis of Compound 54a:1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinoline-4,8-diamine

Compound 54 (10 mg, 0.021 mmol) was dissolved in 1 mL of HCl/dioxanesolution and stirred for 12 hours. The solvent was then removed undervacuum and the residue was washed with diethyl ether to obtain thecompound 54a in quantitative yields. ¹H NMR (500 MHz, MeOD) δ 8.10 (s,1H), 7.97 (d, J=8.9 Hz, 1H), 7.67 (dd, J=8.9, 2.2 Hz, 1H), 7.51 (d,J=8.2 Hz, 2H), 7.23 (d, J=8.1 Hz, 2H), 6.04 (s, 2H), 4.12 (s, 2H), 3.03(t, J=7.6 Hz, 2H), 1.92-1.84 (m, 2H), 1.53-1.43 (m, 2H), 0.96 (t, J=7.4Hz, 3H). ¹³C NMR (126 MHz, DMSO) b 156.84, 147.83, 135.56, 134.67,133.58, 129.58, 129.54, 126.08, 125.63, 124.97, 119.52, 113.08, 48.08,41.64, 29.09, 26.17, 21.75, 13.66. MS (ESI) calculated for C₂₂H₂₆N₆, m/z374.2219, found 375.2508 (M+H)+.

Synthesis of Compound 55b:N¹,N⁸-bis(4-amino-1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)octanediamide

To a solution of 54 (74 mg, 0.156 mmol) in anhydrous THF, were addedtriethylamine (39 mg, 0.39 mmol) and suberoyl chloride (15 mg, 0.07mmol), and the reaction mixture was stirred for 1 hour. The solvent wasthen removed under vacuum and the residue was purified using columnchromatography (20% MeOH/dichloromethane) to obtain the bis-N-Bocprotected compound which was then dissolved in 1 mL of HCl/dioxanesolution and stirred for 14 hours.

The solvent was then removed under vacuum and the residue was washedwith diethyl ether to afford the compound 55b (12 mg, 19%; low yieldswere due to partial acylation of the C4-NH₂ which was found to beunstable). ¹H NMR (500 MHz, MeOD) δ 8.62 (s, 2H), 7.70-7.65 (m, 2H),7.61 (d, J=9.0 Hz, 2H), 7.44 (d, J=7.9 Hz, 4H), 7.18 (d, J=7.7 Hz, 4H),5.93 (s, 4H), 4.07 (s, 4H), 2.98 (t, J=7.5 Hz, 4H), 2.37 (dd, J=16.6,9.4 Hz, 4H), 1.85 (dt, J=15.0, 7.6 Hz, 4H), 1.71 (s, 4H), 1.52-1.40 (m,8H), 0.94 (t, J=7.3 Hz, 6H). ¹³C NMR (126 MHz, MeOD) b 174.75, 159.05,149.75, 137.64, 137.34, 137.11, 134.51, 131.24, 130.93, 127.87, 126.22,123.18, 119.97, 114.28, 111.99, 49.82, 43.82, 37.93, 30.27, 30.01,27.81, 26.63, 23.36, 14.11. MS (ESI) calculated for C₅₂H₆₂N₁₂O₂, m/z886.5119, found 909.5031 (M+Na⁺) and 444.2632 (M+2H)²⁺.

Compounds 55a and 55c were synthesized similarly as described forcompound 55b.

55a:N¹,N⁶-bis(4-amino-1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)adipamide

¹H NMR (500 MHz, MeOD) δ 8.57 (s, 2H), 7.62-7.54 (m, 4H), 7.35 (d, J=8.1Hz, 4H), 7.10 (d, J=8.0 Hz, 4H), 5.85 (s, 4H), 3.98 (s, 4H), 2.90 (t,J=7.6 Hz, 4H), 2.36 (s, 4H), 1.77 (dt, J=15.2, 7.6 Hz, 4H), 1.73-1.61(m, 4H), 1.45-1.30 (m, 4H), 0.86 (t, J=7.4 Hz, 6H). ¹³C NMR (126 MHz,MeOD) δ 174.43, 159.11, 149.83, 137.66, 137.40, 137.16, 134.53, 131.33,130.94, 127.89, 126.30, 123.18, 120.01, 114.38, 112.05, 49.82, 43.83,37.65, 30.32, 27.81, 26.28, 23.37, 14.12. MS (ESI) calculated forC₅₀H₅₈N₁₂O₂, m/z 858.4806, found 859.4131 (M+H)⁺ and 430.2113 (M+2H)²⁺.

55c:N¹,N¹²-bis(4-amino-1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)dodecanediamide

¹H NMR (500 MHz, MeOD) δ 8.63 (s, 2H), 7.66 (d, J=9.0 Hz, 2H), 7.58 (d,J=8.9 Hz, 2H), 7.41 (d, J=7.9 Hz, 4H), 7.15 (d, J=7.8 Hz, 4H), 5.91 (s,4H), 4.04 (s, 4H), 2.96 (t, J=7.5 Hz, 4H), 2.33 (t, J=7.1 Hz, 4H), 1.83(dt, J=15.2, 7.7 Hz, 4H), 1.65 (s, 4H), 1.44 (dq, J=14.7, 7.3 Hz, 4H),1.38-1.21 (m, 12H), 0.92 (t, J=7.3 Hz, 6H). ¹³C NMR (126 MHz, MeOD) b174.82, 159.01, 149.63, 137.68, 137.27, 137.10, 134.50, 131.24, 130.93,129.84, 127.86, 126.00, 123.16, 120.01, 114.24, 111.92, 49.87, 43.82,43.75, 38.04, 30.45, 30.34, 30.29, 30.23, 27.78, 26.78, 23.35, 14.11. MS(ESI) calculated for C₅₆H₇₀N₁₂O₂, m/z 942.5745, found 943.5746 (M+H)⁺and 472.2987 (M+2H)²⁺.

TLR3/7/8 Reporter Gene Assays (NF-κB Induction):

The induction of NF-κB was quantified using HEK-Blue-3, HEK-Blue-7 andHEK-Blue-8 cells as previously described by us.^(8,24,51) HEK293 cellswere stably transfected with human TLR3 (or human TLR7 or human TLR8),MD2, and secreted alkaline phosphatase (sAP), and were maintained inHEK-Blue™ Selection medium containing zeocin and normocin. Stableexpression of secreted alkaline phosphatase (sAP) under control ofNF-κB/AP-1 promoters is inducible by the TLR3 (or TLR7 or TLR8)agonists, and extracellular sAP in the supernatant is proportional toNF-κB induction. HEK-Blue cells were incubated at a density of ˜105cells/ml in a volume of 80 μl/well, in 384-well, flat-bottomed, cellculture-treated microtiter plates until confluency was achieved, andsubsequently graded concentrations of stimuli. sAP was assayedspectrophotometrically using an alkaline phosphatase-specific chromogen(present in HEK-detection medium as supplied by the vendor) at 620 nm.

Antagonism assays were done as described by us earlier⁷³ using thefollowing agonists at a constant concentration: TLR3 Poly(I:C) (10ng/mL); TLR7: gardiquimod (1 μg/mL); TLR8: CL075 (1 μg/mL) mixed withgraded concentrations of the test compounds.

IFN-α Induction in Human PBMCs:

Aliquots (10⁶ cells in 100 μL) of hPBMCs isolated from blood obtainedfrom healthy human donors after informed consent by conventionalFicoll-Hypaque gradient centrifugation were stimulated for 12 h withgraded concentrations of test compounds. The supernatant was isolated bycentrifugation, diluted 1:20, and IFN-α was assayed in triplicate usinga high-sensitivity human IFN-α-specific ELISA kit (PBL InterferonSource, Piscataway, N.J.).

Cytokine and Chemokine in Human PBMCs:

Aliquots (10⁶ cells in 100 μL) of hPBMCs isolated from blood obtainedfrom healthy human donors after informed consent by conventionalFicoll-Hypaque gradient centrifugation were stimulated for 12 h withgraded concentrations of test compounds. The supernatant was isolated bycentrifugation, diluted 1:20, and cytokines and chemokines were assayedin triplicate using analyte-specific cytokine/chemokine bead arrayassays as reported by us previously.⁷⁴

With the exception of the 47 and 49 series of compounds, all otherdimers retained TLR7-agonistic properties, the 52 series being the mostpotent (FIG. 19A); interestingly, only 52c displayed both TLR7 and TLR8agonism (FIGS. 19A and 19B, respectively, and Table 1), suggestingadvantages of a long linker for dual agonism. The C2-linked dimericcompounds 47a, 47b, 49a, and 49b unexpectedly showed potent antagonisticactivity in both TLR7 and TLR8 assays, with 47a being most potent (IC₅₀values of 3.1 and 3.2 μM in TLR7 and TLR8 assays, FIGS. 20A and 20B,respectively; Table 1).

Both agonistic and antagonistic compounds were then tested inappropriate secondary screens employing ex vivo human blood-derivedmodels. The ligation of TLR7 and TLR8 trigger inflammatory responsescharacterized by the elaboration of type I interferon (IFN-α/β) byvirus-infected cells via activation of downstream NF-κB and IFN-3promoters.⁷⁵⁻⁸⁰ IFN production is a hallmark response underlyingcellular antiviral immune responses. It was desirable to verify that theTLR7 agonism observed (FIG. 19) manifested in IFN production insecondary screens. Using an ex vivo stimulation model using humanperipheral blood mononuclear cells (hPBMC), it was demonstrated thatIFN-α was indeed induced in a dose-dependent, bimodal manner as expectedfor innate immune responses (FIG. 21). Compound 52c was found to be themost potent.

The antagonistic properties of 47a in inhibiting TLR7 and TLR8-mediatedinduction of various proinflammatory cytokines (FIGS. 22A-22H) andchemokines (FIGS. 23A-23D) were examined in detail in ex vivo modelsusing human blood, since this compound was found to be the most potentantagonist in the series in primary screens (Table 1). The potency of47a was compared alongside chloroquine, which is known to selectivelysuppress intracellular TLR7, but not TLR8 signaling via inhibition ofendolysosomal acidification.^(81,82) We found 47a to be a potentinhibitor of both TLR7 and TLR8-induced cytokine and chemokine release,with IC₅₀ values of about 0.05-0.3 μM (FIGS. 22, 23). TLR8 signalingmanifests predominantly in the induction of pro-inflammatory cytokinessuch as TNF-α and IL-113.^(83,84) Chloroquine, a TLR7 antagonist, is afeeble inhibitor of TNF-α and IL-1β, while 47a, as would be expected fora TLR8 antagonist, potently inhibits the production of theseproinflammatory cytokines (FIG. 23), as well as IL-6 and IL-8 which aretypically induced secondarily, in an autocrine/paracrine manner. Therelative specificity of chloroquine in inhibiting TLR7 as well as thedual TLR7/8-inhibitory activities of 47a are also evident in Schildplots (FIG. 24A-24B). Although the relationship between antagonistconcentration and change in EC₅₀ for TLR7 inhibition by 47a isnear-ideal (slope: 1.12, FIG. 24A), a distinct deviation from idealcompetitive inhibition for TLR8 is observed (slope: 0.51, FIG. 24B),suggesting that additional mechanisms for TLR8 inhibition, possiblyallosteric, may be operational.

TABLE 1 Agonistic and antagonistic activities of the dimers in TLR7 andTLR8 reporter gene assays. Com- TLR7 TLR8 pound Antag- Antag- num-Agonism onism Agonism onism ber Structure (μM) (μM) (μM) (μM) 47a

ND 3.1 ND 3.2 47b

ND ND ND 15.63 49a

ND ND ND 10.92 49b

ND 17.88 ND 4.65 51a

2.05 ND ND ND 51b

0.56 ND ND ND 51c

3.00 ND ND ND 51d

1.42 ND ND ND 52a

0.11 ND ND ND 52b

0.24 ND ND ND 52c

0.17 ND 4.78 ND 53a

0.56 ND ND ND 54a

0.45 ND ND ND 55a

7.24 ND ND ND 55b

4.02 ND ND ND 55c

5.4  ND ND ND ND = not detected; NT = not tested.

In conclusion, the present data demonstrate that the C4, C8, andN¹-aryl-linked dimers are agonists, with the last being most potent. TheN¹-aryl-linked dimers are of interest as potential vaccine adjuvants arecurrently being evaluated in animal models. The C2-linked dimers werefound to be potently antagonistic at both TLR7 and TLR8 and may beuseful as small molecule probes for examining the effects of inhibitingendolysosomal TLR signaling in HIV and autoimmune states.

Example 9

Dual TLR2/TLR7 Adjuvants

Toll-like receptor 2-agonistic lipopeptides typified byS-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cysteinyl-S-serine (PAM(2)CS)compounds are potential vaccine adjuvants.^(52,85,86) Combining theindividual TLR-agonistic activities of the PAM2CS (TLR2) andimidazoquinoline (TLR7) chemotypes may result in highly potentadjuvants. Six different hybrids in various configurations (60-65) weresynthesized to examine how the TLR2 and TLR7 activities may be modulatedin such compounds, and how such differences could manifest in adjuvanticactivities in vivo. Imidazoquinoline derived compounds incorporating afree amine, carboxylic acid, or an isothiocyanate, with or without atriethylene glycol spacer were synthesized (Scheme 2), as were analoguesof PAM2CS with a free carboxylate on the serine or a free amine on thecysteine fragment of the lipopeptide (Scheme 19). Coupling thesesynthons yielded six differently configured hybrids (Schemes 20-22).These analogues show divergent TLR2- and TLR7-specific activities inprimary screens (FIGS. 25A and 25B, respectively), as well as insecondary cytokine induction screens (FIGS. 26A-26E). These hybrids arealso adjuvantic in rabbit immunization studies (FIG. 27).

Compounds 56, 58 and 59 were synthesized as describedearlier.^(52,85,86)

Synthesis of Compound 57:(S)-2-((R)-3-(((R)-2,3-bis(palmitoyloxy)propyl)thio)-2-((tert-butoxycarbonyl)amino)propanamido)-3-hydroxypropanoicacid

Compound 56 (200 mg, 0.21 mmol) was dissolved in trifluoroacetic acidand stirred for 40 min, followed by removal of the solvent under vacuumto obtain the free amine intermediate (170 mg), which was then dissolvedin anhydrous CH₂Cl₂ followed by the addition of triethylamine (53 mL,0.38 mmol) and di-tert-butyl dicarbonate (46 mg, 0.21 mmol). Thereaction mixture was stirred for 2 hours followed by removal of thesolvent under vacuum. The residue was purified using columnchromatography (6% MeOH/CH₂Cl₂) to obtain the compound 57 (100 mg, 57%).MS (ESI) calculated for C₄₆H₈₆N₂O₁₀S, m/z 858.6003, found 859.6112(M+H)⁺.

Synthesis of Compound 60

To the solution of compound 8 (50 mg, 0.12 mmol) in anhydrous pyridinewas added compound 59 (166 mg, 0.19 mmol). The reaction mixture washeated at 45° C. for 24 hours, followed by removal of the solvent undervacuum. The residue was then purified using column chromatography toobtain the compound 60 (53 mg, 38%). ¹H NMR (500 MHz, CDCl₃) δ 15.40 (d,J=18.5 Hz, 1H), 10.56 (d, J=17.8 Hz, 1H), 7.81 (d, J=8.3 Hz, 1H), 7.68(d, J=8.3 Hz, 1H), 7.47 (t, J=7.7 Hz, 1H), 7.35-7.28 (m, 3H), 7.28-7.23(m, 1H), 6.98 (d, J=6.6 Hz, 2H), 5.72 (s, 2H), 5.37-5.07 (m, 2H), 4.83(s, 1H), 4.71-4.53 (m, 2H), 4.33 (dd, J=11.7, 2.7 Hz, 1H), 4.08 (ddd,J=27.8, 11.9, 6.5 Hz, 1H), 4.00-3.88 (m, 2H), 3.71 (d, J=6.8 Hz, 3H),3.26-3.01 (m, 4H), 2.91-2.77 (m, 4H), 2.73 (d, J=6.4 Hz, 1H), 2.34-2.25(m, 4H), 1.84-1.76 (m, 2H), 1.56 (s, 4H), 1.49-1.39 (m, 2H), 1.33-1.19(m, 48H), 0.94 (t, J=7.3 Hz, 3H), 0.87 (t, J=6.9 Hz, 6H). ¹³C NMR (126MHz, CDCl₃) δ 174.58, 174.34, 173.83, 173.78, 170.50, 170.14, 156.77,156.75, 149.65, 149.63, 135.60, 134.58, 132.97, 129.66, 128.68, 128.66,125.63, 125.18, 124.72, 124.70, 120.56, 119.66, 112.45, 71.40, 70.26,63.99, 63.81, 62.56, 62.40, 57.55, 57.35, 55.19, 55.02, 52.72, 52.69,48.97, 47.80, 35.62, 34.47, 34.37, 34.23, 34.09, 33.91, 32.76, 31.93,29.72, 29.69, 29.67, 29.52, 29.51, 29.37, 29.32, 29.29, 29.12, 29.09,29.08, 26.93, 24.90, 24.88, 24.84, 24.83, 22.70, 22.38, 14.13, 13.72. MS(ESI) calculated for C₆₅H₁₀₃N₇O₈S₂, m/z 1173.7310, found 1174.7410(M+H)⁺.

Synthesis of Compound 61

To the solution of compound 15 (50 mg, 0.07 mmol) in anhydrous pyridinewas added compound 59 (54 mg, 0.07 mmol). The reaction mixture washeated at 45° C. for 24 hours, followed by removal of the solvent undervacuum. The residue was then purified using column chromatography (12%MeOH/CH₂Cl₂) to obtain the compound 61 (55 mg, 53%). ¹H NMR (500 MHz,MeOD) δ 7.84 (d, J=8.2 Hz, 1H), 7.68 (d, J=8.0 Hz, 1H), 7.48-7.44 (m,1H), 7.26 (d, J=8.2 Hz, 2H), 7.18-7.14 (m, 1H), 7.04 (d, J=8.2 Hz, 2H),5.87 (s, 2H), 5.23 (bs, 2H), 4.53 (t, J=4.4 Hz, 1H), 4.38 (dd, J=11.9,3.0 Hz, 1H), 4.33 (s, 2H), 4.14 (dd, J=12.0, 6.5 Hz, 1H), 3.91 (dd,J=11.3, 4.6 Hz, 1H), 3.80 (dd, J=11.3, 4.1 Hz, 1H), 3.73 (s, 3H), 3.61(dd, J=5.9, 3.0 Hz, 4H), 3.56 (dd, J=5.7, 2.7 Hz, 4H), 3.53-3.47 (m,4H), 3.22 (t, J=6.8 Hz, 2H), 3.14-3.07 (m, 1H), 3.04-2.92 (m, 3H), 2.88(dd, J=14.1, 6.1 Hz, 1H), 2.80 (dd, J=14.2, 7.3 Hz, 1H), 2.35-2.28 (m,4H), 2.21 (dt, J=21.8, 7.4 Hz, 4H), 1.91-1.76 (m, 6H), 1.72 (p, J=6.5Hz, 2H), 1.64-1.53 (m, 4H), 1.44 (dt, J=14.7, 7.4 Hz, 2H), 1.27 (s,50H), 0.94 (t, J=7.4 Hz, 3H), 0.89 (t, J=7.0 Hz, 6H). ¹³C NMR (126 MHz,MeOD) δ 175.24, 174.95, 174.76, 171.98, 156.76, 152.19, 140.07, 136.02,135.90, 129.48, 129.10, 126.94, 126.79, 124.11, 121.90, 115.50, 79.56,79.30, 79.04, 71.97, 71.54, 71.52, 71.20, 71.15, 69.97, 65.02, 62.83,56.34, 56.29, 52.95, 49.72, 43.66, 37.92, 36.34, 36.21, 35.26, 35.02,33.72, 33.13, 30.88, 30.84, 30.81, 30.73, 30.71, 30.55, 30.52, 30.33,30.26, 30.24, 27.89, 26.12, 26.09, 23.80, 23.47, 23.28, 14.55, 14.21. MS(ESI) calculated for C₈₀H₁₃₁N₉O₁₃S₂, m/z 1489.9308, found 1490.9570(M+H)⁺.

Synthesis of Compound 62

To a solution of compound 57 (60 mg, 0.07 mmol) in anhydrous DMF, wereadded triethylamine (24 μL, 0.18 mmol), HBTU (29 mg, 0.08 mmol) and 7d(30 mg, 0.07 mmol). The reaction mixture was stirred for 8 hoursfollowed by removal of the solvent under vacuum to obtain the residuewhich was purified using column chromatography (8% MeOH/CH₂Cl₂) toobtain the compound 62 (26 mg, 34%). ¹H NMR (500 MHz, CDCl₃) δ 10.79(bs, 1H), 8.36 (d, J=37.2 Hz, 1H), 7.88 (t, 1H), 7.68 (t, 1H), 7.48 (t,J=5.7 Hz, 1H), 7.39 (s, 1H), 7.24 (d, J=6.7 Hz, 1H), 6.97 (d, J=7.0 Hz,1H), 5.72 (s, 1H), 5.11 (s, 1H), 4.47-4.38 (m, 1H), 4.33 (d, J=9.5 Hz,1H), 4.15-4.02 (m, 1H), 3.73-3.60 (m, 1H), 3.00 (s, 1H), 2.90-2.82 (m,1H), 2.73-2.63 (m, 1H), 2.33-2.26 (m, 1H), 1.86-1.76 (m, 1H), 1.59 (s,1H), 1.44 (dd, J=14.5, 7.3 Hz, 1H), 1.34-1.18 (m, 8H), 0.94 (t, J=7.2Hz, 1H), 0.88 (t, J=6.9 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 173.56,173.45, 170.77, 156.66, 149.84, 138.59, 135.50, 134.73, 133.03, 129.56,128.51, 125.64, 125.05, 124.96, 120.46, 119.84, 112.54, 70.25, 63.61,62.30, 54.03, 48.89, 42.72, 34.29, 34.08, 32.77, 31.93, 29.72, 29.69,29.67, 29.52, 29.37, 29.31, 29.14, 29.12, 26.96, 24.92, 24.86, 22.70,22.38, 14.13, 13.72. MS (ESI) calculated for C₆₃H₁₀₁N₇O₇S, m/z1099.7483, found 1100.7603 (M+H)⁺.

Synthesis of Compound 63

To a solution of compound 57 (40 mg, 0.05 mmol) in anhydrous DMF, wereadded triethylamine (16 μL, 0.12 mmol), HBTU (20 mg, 0.05 mmol) and 15(37 mg, 0.05 mmol). The reaction mixture was stirred for 8 hoursfollowed by removal of the solvent under vacuum to obtain the residuewhich was purified using column chromatography (10% MeOH/CH₂Cl₂) toobtain the compound 63 (16 mg, 24%). ¹H NMR (500 MHz, CDCl₃) δ 15.18 (s,1H), 10.33 (s, 1H), 9.05 (s, 1H), 7.89-7.58 (m, 3H), 7.58-7.33 (m, 2H),7.34-7.15 (m, 2H), 7.03-6.82 (m, 2H), 5.98 (s, 1H), 5.71 (s, 2H), 5.14(s, 1H), 4.45-4.25 (m, 5H), 4.06 (s, 2H), 3.90-3.35 (m, 18H), 3.30-2.64(m, 12H), 2.37-2.10 (m, 7H), 2.03 (s, 3H), 1.86-1.50 (m, 11H), 1.41 (s,2H), 1.24 (s, 44H), 0.95-0.83 (m, 9H). ¹³C NMR (126 MHz, CDCl₃) δ173.68, 156.97, 149.61, 138.93, 135.59, 134.44, 132.80, 128.69, 125.55,125.16, 120.70, 112.48, 69.88, 69.39, 63.72, 48.94, 35.14, 34.25, 34.03,31.93, 29.73, 29.68, 29.56, 29.38, 29.16, 26.85, 24.90, 24.84, 22.69,22.30, 14.13, 13.63. MS (ESI) calculated for C₇₈H₁₂₉N₉O₁₂S, m/z1415.9481, found 1438.9406 (M+Na⁺).

Synthesis of Compound 64

To a solution of compound 13 (50 mg, 0.09 mmol) in anhydrous DMF, wereadded triethylamine (30 μL, 0.22 mmol), HBTU (36 mg, 0.10 mmol) and 59(77 mg, 0.09 mmol). The reaction mixture was stirred for 8 hoursfollowed by removal of the solvent under vacuum to obtain the residuewhich was purified using column chromatography (6% MeOH/CH₂Cl₂) toobtain the compound 64 (40 mg, 33%). ¹H NMR (500 MHz, CDCl₃) δ 7.92 (bs,2H), 7.75 (d, J=8.1 Hz, 1H), 7.69 (t, J=8.3 Hz, 2H), 7.65-7.60 (m, 1H),7.36 (dd, J=14.2, 7.0 Hz, 1H), 7.32-7.24 (m, 1H), 7.23-7.16 (m, 7H),7.06-6.98 (m, 1H), 6.94 (dd, J=17.3, 6.3 Hz, 2H), 5.67 (s, 2H),5.18-5.08 (m, 1H), 4.64-4.50 (m, 2H), 4.36-4.27 (m, 3H), 4.10 (dd,J=11.9, 6.3 Hz, 1H), 3.97-3.84 (m, 3H), 3.62 (d, J=8.7 Hz, 3H),3.02-2.87 (m, 2H), 2.86-2.79 (m, 2H), 2.77-2.66 (m, 2H), 2.34-2.19 (m,8H), 1.91 (s, 2H), 1.76 (dt, J=14.1, 7.1 Hz, 2H), 1.63-1.53 (m, 4H),1.39 (dd, J=14.1, 6.7 Hz, 2H), 1.33-1.15 (m, 48H), 0.88 (dt, J=12.4, 4.4Hz, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 173.92, 173.86, 173.73, 173.69,173.65, 173.54, 170.96, 170.80, 170.67, 157.21, 148.82, 142.53, 138.97,135.91, 134.09, 132.80, 129.71, 128.66, 127.96, 125.64, 125.53, 125.46,124.99, 124.61, 120.92, 119.35, 118.13, 112.70, 111.14, 70.35, 70.22,63.72, 62.22, 55.04, 53.05, 52.66, 52.63, 52.44, 48.88, 42.83, 34.93,34.75, 34.36, 34.21, 34.10, 33.57, 32.88, 32.02, 31.93, 29.72, 29.67,29.55, 29.53, 29.37, 29.32, 29.16, 29.13, 26.85, 24.93, 24.89, 24.86,22.70, 22.29, 21.55, 21.42, 14.14, 13.69. MS (ESI) calculated forC₆₉H₁₀₉N₇O₁₀S, m/z 1227.7957, found 1228.8095 (M+H)⁺.

Synthesis of Compound 65

To a solution of compound 12 (37 mg, 0.05 mmol) in anhydrous DMF, wereadded triethylamine (16 μL, 0.12 mmol), HBTU (20 mg, 0.05 mmol) and 59(42 mg, 0.05 mmol). The reaction mixture was stirred for 8 hoursfollowed by removal of the solvent under vacuum to obtain the residuewhich was purified using column chromatography (18% MeOH/CH₂Cl₂) toobtain the compound 65 (20 mg, 28%). ¹H NMR (500 MHz, CDCl₃) δ 7.82 (dd,J=15.3, 7.9 Hz, 1H), 7.70 (d, J=8.2 Hz, 1H), 7.45 (t, J=7.7 Hz, 1H),7.24 (d, J=8.0 Hz, 1H), 7.15 (t, J=7.6 Hz, 1H), 6.98 (d, J=8.0 Hz, 1H),6.70 (dd, J=11.6, 5.9 Hz, 1H), 5.71 (s, 1H), 5.15 (ddd, J=9.5, 6.3, 3.1Hz, 1H), 4.75-4.67 (m, 1H), 4.58 (ddd, J=13.5, 7.2, 3.5 Hz, 1H), 4.38(d, J=5.8 Hz, 1H), 4.32 (ddd, J=10.4, 7.2, 3.2 Hz, 1H), 4.15-4.09 (m,1H), 4.05-3.94 (m, 1H), 3.90 (td, J=11.4, 2.8 Hz, 1H), 3.76-3.70 (m,2H), 3.65-3.45 (m, 7H), 3.34-3.20 (m, 3H), 3.07 (dd, J=13.9, 6.9 Hz,1H), 3.01-2.93 (m, 1H), 2.90-2.86 (m, 1H), 2.77-2.69 (m, 1H), 2.35-2.22(m, 5H), 2.21-2.16 (m, 2H), 2.00-1.77 (m, 4H), 1.74-1.67 (m, 2H),1.63-1.57 (m, 2H), 1.49-1.40 (m, 1H), 1.31-1.22 (m, 26H), 0.94 (t, J=7.4Hz, 2H), 0.88 (t, J=6.9 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 173.60,173.54, 173.52, 173.32, 173.26, 173.13, 172.96, 172.86, 170.92, 170.68,170.64, 170.56, 154.64, 150.89, 138.75, 134.26, 134.02, 128.55, 127.71,126.28, 125.73, 122.92, 119.92, 114.54, 70.37, 70.30, 70.08, 69.87,69.85, 69.83, 69.78, 69.51, 63.71, 62.46, 62.29, 55.27, 52.66, 52.54,52.07, 48.68, 42.84, 37.74, 37.70, 37.56, 37.47, 35.47, 35.29, 35.05,34.99, 34.75, 34.34, 34.10, 33.82, 33.11, 32.24, 31.93, 29.84, 29.72,29.69, 29.67, 29.54, 29.52, 29.37, 29.32, 29.15, 29.13, 28.91, 28.89,28.85, 27.10, 24.94, 24.91, 24.87, 22.70, 22.50, 22.02, 21.61, 21.48,14.14, 13.79. MS (ESI) calculated for C₈₄H₁₃₇N₉O₁₅S, m/z 1543.9955,found 1545.0105 (M+H)⁺.

REFERENCES

The following references are hereby incorporated herein in theirentirety.

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1. An imidazoquinoline derived compound of Formula I, a derivativethereof, or analogue thereof, or pharmaceutically acceptable saltthereof, wherein Formula I has the structure:

wherein, R is selected from the group consisting of: —NH(R₅) andisothiocyanate; R₅ is selected from the group consisting of hydrogen,acetyl, —CO-tert-Bu (-Boc), —CO—(CH₂)_(x)—R₆, C₁-C₁₆ alkyl,—CO-4-(phenylboronic acid),—C(S)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH₂,

 a reporter moiety, a tissue-specific moiety, a peptide antigen moiety,a protein antigen moiety, a polysaccharide antigen moiety, and a TLR2agonist moiety; R₆ is selected from the group consisting of hydrogen,alkyne, azido, carboxylic acid, and—CONH—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—R₇; R₇ is selectedfrom the group consisting of amino, isothiocyanate, and—NH—CO—(CH₂)_(x)—CO₂H; R₈ is selected from a peptide antigen moiety or aprotein antigen moiety; and x is any integer from 1 to
 10. 2. Theimidazoquinoline derived compound of claim 1, wherein the compound iscapable of activating Toll-like receptor (TLR)
 7. 3. Theimidazoquinoline derived compound of claim 1, wherein the compound iscapable of activating TLR7 and TLR8.
 4. The imidazoquinoline derivedcompound of claim 1, wherein the compound of Formula I is chosen from anisothiocyanate derivative of Formula I and a maleimide derivative ofFormula I.
 5. The imidazoquinoline derived compound of claim 1, whereinR₅ comprises a reporter moiety comprising a reporter molecule capable ofproducing a detectable signal.
 6. The imidazoquinoline derived compoundof claim 1, wherein R₅ comprises an antigen moiety is chosen from apeptide antigen moiety, a protein antigen moiety, or a polysaccharideantigen moiety, and wherein the imidazoquinoline derived compound iscapable of activating TLR7.
 7. The imidazoquinoline derived compound ofclaim 1, wherein R₅ comprises a tissue-specific moiety including atissue-specific agent.
 8. The imidazoquinoline derived compound of claim1, wherein R₅ comprises a TLR2 agonist moiety including a TLR2 agonist,and wherein the imidazoquinoline derived compound is capable of dualactivation of TLR2 and TLR7.
 9. An imidazoquinoline derived compoundcomprising a dimer or a dendrimer of a compound of Formula I, a compoundof Formula II, derivatives thereof, analogues thereof, orpharmaceutically acceptable salts thereof, wherein

wherein, R₁ and R₃ are each independently selected from the groupconsisting of hydrogen, halogen, nitro, —NH₂, azido, hydroxyl, —CF₃,carboxylic acid and —CO₂R₂; R₂ is a C₂-C₅ alkyl, and R for Formula I andR₄ for Formula II are each independently selected from the groupconsisting of: —NH(R₅) and isothiocyanate; R₅ is selected from the groupconsisting of hydrogen, acetyl, —CO-tert-Bu (-Boc), —CO—(CH₂)_(x)—R₆,C₁-C₁₆ alkyl, —CO-4-(phenylboronic acid),—C(S)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH₂,

a reporter moiety, a tissue-specific moiety, a peptide antigen moiety, aprotein antigen moiety, a polysaccharide antigen moiety, and a TLR2agonist moiety; R₆ is selected from the group consisting of hydrogen,alkyne, azido, carboxylic acid, and—CONH—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—R₇; R₇ is selectedfrom the group consisting of amino, isothiocyanate, and—NH—CO—(CH₂)_(x)—CO₂H; R₈ is selected from a peptide antigen moiety or aprotein antigen moiety; and x is any integer from 1 to
 10. 10. Theimidazoquinoline derived compound of claim 22, wherein the dimer ordendrimer compound is a Toll-like receptor (TLR) 7 agonist or a dualTLR7/TLR8 agonist.
 11. An imidazoquinoline derived compound of FormulaII, a derivative thereof, analogue thereof, or pharmaceuticallyacceptable salt thereof, wherein Formula II has the structure:

wherein, R₁ and R₃ are each independently selected from the groupconsisting of hydrogen, halogen, nitro, —NH₂, azido, hydroxyl, —CF₃,carboxylic acid, and —CO₂R₂; R₂ is a C₂-C₅ alkyl, and R₄ selected fromthe group consisting of: —NH(R₅) and isothiocyanate; R₅ is selected fromthe group consisting of hydrogen, acetyl, —CO-tert-Bu (-Boc),—CO—(CH₂)_(x)—R₆, C₁-C₁₆ alkyl, —CO-4-,—C(S)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH—(CH₂)_(x)—NH₂,

 a reporter moiety, a tissue-specific moiety, a peptide antigen moiety,a protein antigen moiety, a polysaccharide antigen moiety, and a TLR2agonist moiety; R₆ is selected from the group consisting of hydrogen,alkyne, azido, carboxylic acid, and—CONH—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—O—(CH₂)_(x)—R₇; R₇ is selectedfrom the group consisting of amino, isothiocyanate, and—NH—CO—(CH₂)_(x)—CO₂H; R₈ is selected from a peptide antigen moiety or aprotein antigen moiety; and x is any integer from 1 to
 10. 12. Theimidazoquinoline derived compound of claim 31, wherein the compound iscapable of activating TLR7 or dual activation of TLR7 and TLR8.